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NEOPLASIA
Expression of the granzyme B inhibitor PI9 predicts outcome in nasal NK/T-cell
lymphoma: results of a Western series of 48 patients treated with first-line
polychemotherapy within the Groupe d’Etude des Lymphomes
de l’Adulte (GELA) trials
Céline Bossard,1 Karim Belhadj,2 Felix Reyes,2 Nadine Martin-Garcia,1 Françoise Berger,3 Jean Alain Kummer,4
Josette Brière,5 Anne-Catherine Baglin,6 Stéphane Cheze,7 Jacques Bosq,8 Vincent Ribrag,9 Christian Gisselbrecht,10
Nicolas Mounier,10 and Philippe Gaulard1
1Département
de Pathologie and Institut National de la Santé et de la Recherche Médicale (Inserm) Unité (U) 617, Hôpital Henri Mondor, Assistance
Publique-Hôpitaux de Paris (AP-HP), Créteil, France; 2Service d’Hématologie Clinique, Hôpital Henri Mondor, Créteil, France; 3Service d’Anatomie et de
Cytologie Pathologiques, Centre Hospitalier Lyon Sud, Pierre Bénite, France; 4Department of Pathology, University Medical Center Utrecht, The Netherlands;
5Département de Pathologie, Hôpital Saint-Louis, Paris, France; 6Département de Pathologie, Hôpital Foch, Suresnes, France; 7Service d’Hématologie Clinique,
Hôpital Clemenceau, Caen, France; 8Département de Pathologie, Institut Gustave Roussy, Villejuif, France; 9Département de Médecine, Institut Gustave
Roussy, Villejuif, France; 10Institut d’Hématologie, Hôpital Saint-Louis, Paris, France
Nasal NK/T-cell lymphoma is a rare disease entity with a poor outcome. Expression of antiapoptotic proteins has not
been extensively investigated in this entity. Forty-eight patients with nasal T/NKcell lymphoma who received first-line
polychemotherapy (n ⴝ 44) or chemoradiotherapy (n ⴝ 4) were analyzed for expression of active caspase-3 (aC3), granzyme B protease inhibitor 9 (PI9), and
Bcl-2 proteins. Lymphomas were CD3ⴙ/
CD5ⴚ/granzyme Bⴙ and EBV-associated.
Median age was 46 years. Stage I/II dis-
ease was present in 75% of the cases and
an International Prognostic Index (IPI)
score less than 1 in 65%. With a median
follow-up of 6.3 years, 5-year event-free
survival (EFS) and overall survival (OS)
rates were 39% and 49%, respectively.
Apoptotic index was scored as high in
32% of cases and PI9 expression as positive in 68%, whereas 35% disclosed a
high number of aC3ⴙ tumor cells. Univariate analysis showed that absence of PI9
and low apoptotic index were associated
with poor outcome, but not aC3 expres-
sion nor IPI score. By multivariate analysis, both parameters affected independently EFS (P ⴝ .02 and .08, respectively)
and OS (P ⴝ .009 and .04). In view of its
constitutive expression by normal NK
cells, it is suggested that loss of PI9
expression in tumor cells may reflect
some mechanism associated with progression. (Blood. 2007;109:2183-2189)
© 2007 by The American Society of Hematology
Introduction
Nasal NK/T-cell lymphoma has been recently recognized as a
distinct lymphoma subtype within the World Health Organization
(WHO) classification.1 It shows a predilection for Asians, Mexicans, and South Americans contrasting with its rarity in Western
countries. Clinically, it is characterized by a localized disease of the
nasal cavity, the nasopharynx, paranasal sinuses, or palate. Nasal
NK/T-cell lymphomas derive putatively from natural killer (NK)
cells or more rarely from a subset of ␥␦ or ␣␤ cytotoxic T cells and
show a striking association with Epstein-Barr virus (EBV).1,2
Treatment strategies have not been fully defined yet,3-11 and most
series, mainly from Eastern countries, have pointed out their poor
clinical outcome after treatment with radiotherapy given alone or in
combination with chemotherapy.
It is generally accepted on the basis of in vitro studies that
the resistance of neoplastic cells to cytotoxic agents may be due
to inhibition of apoptosis,12,13 a programmed cell death activated
by a cascade of proteases called caspases, leading to degradation of vital cytoplasmic and nuclear proteins. Three major
pathways of caspase activation have been described14: (1) a
cell-surface death receptor-signaling pathway (Fas, tumor necrosis factor) leading to activation of caspase-8; (2) a stressinduced pathway defined by a pivotal event—mitochondrial
outer membrane permeabilization—leading to cytochrome c
release and activation of caspase-9; and (3) the cytotoxic
granules pathway used by cytotoxic T lymphocytes (CTLs) and
NK cells against target cells involving the release of cytolytic
enzymes such as perforin and granzyme B (GrB). GrB has been
shown to activate caspase-3 directly or indirectly via the
stress-induced pathway.15 Because the final execution of apoptosis ultimately results in activation of caspase- 3,16 it might be
expected that high levels of activated caspase-3 (aC3) reflect
proper functioning of at least one of the 3 apoptotic pathways
and result in chemotherapy-sensitive tumor cells. In this regard,
it has been reported that a high number of aC3⫹ Reed-Sternberg
cells in pretreatment biopsies predicted a favorable clinical
outcome in Hodgkin lymphoma.17
Submitted July 7, 2006; accepted October 4, 2006. Prepublished online as Blood
First Edition Paper, October 31, 2006; DOI 10.1182/blood-2006-07-033142.
The online version of this article contains a data supplement.
Presented in abstract form at the 46th annual meeting of the American Society
of Hematology, San Diego, CA, December 6, 2004.70
BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2007 by The American Society of Hematology
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BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
BOSSARD et al
Execution of apoptosis also depends on the expression of
apoptosis-regulating proteins, including Bcl-2 and the recently
identified GrB-specific protease inhibitor 9 (PI9). Bcl-2 inhibits
specifically the stress-induced pathway18 and its overexpression
has been shown to be related to poor clinical outcome in diffuse
large B-cell lymphomas.19,20 PI9 is a serine protease, also named
serpin 9, which is present in cytotoxic effector cells to protect them
against their own GrB.21 PI9 has also been shown to be expressed
in the neoplastic cells of several lymphoma subtypes, including
NK/T-cell lymphoma, suggesting a novel mechanism for tumor
cells to escape their elimination by CTLs.22 In this respect, high
numbers of PI9⫹ neoplastic cells have been reported to predict a
poor outcome in anaplastic large cell lymphoma, whereas a high
number of aC3⫹ tumor cells correlated with a more favorable
prognosis.23
This prompted us to analyze a series of 48 patients with nasal
Tl/NK-cell lymphoma who received chemotherapy as first-line
treatment according to Groupe d’Etude des Lymphomes de l’Adulte
(GELA) protocols and specially to investigate in pretreatment
biopsies whether activation of caspase-3 and expression levels
of the antiapoptotic Bcl-2 and PI9 proteins were related to
clinical outcome.
Patients, materials, and methods
Patient selection
Forty-eight patients with proven nasal NK/T-cell lymphoma diagnosed
between 1987 and 2003 were selected from the files of the LNH 87, 93, and
98 GELA protocols (45 patients) and from the files of the Department of
Pathology, Hôpital Henri Mondor, Créteil, France (3 patients) on the basis
of the following criteria: (1) diagnosis of NK/T-cell lymphoma in a primary
biopsy of the nasopharyngeal region (excluding Waldeyer ring) and (2)
clinical and follow-up data available. In this study, we required for a
diagnosis of nasal NK/T-cell lymphoma that the malignant cells had a
CD3⑀⫹ CD20⫺ phenotype, a cytotoxic profile, and disclosed association
with EBV. According to French legislation at the time of the study, no
informed consent was required to perform supplemental immunohistochemical studies on archival material. The LNH 87, LNH 93, and LNH 98 trials
from the GELA are institutional review board–approved protocols, approved by the Hôpital Saint-Louis (Paris, France).
Response to treatment was considered as complete (CR), unconfirmed
complete (CRu), or failure according to the International Workshop
criteria.35
Histopathologic studies
At the time of enrollment in the LNH87, LNH93, and LNH98 protocols,
tumor biopsies were reviewed and initially classified according to the
updated Kiel classification and Working Formulation,36,37 based on morphologic examination of slides stained with hematoxylin and eosin or Giemsa
and on immunohistochemistry comprising at least CD20 and CD3⑀. For the
purpose of the present study, 2 hematopathologists (P.G. and C.B.)
reviewed all CD3⫹ lymphomas diagnosed on a nasopharyngeal biopsy and
completed the phenotype to reclassify them according to the WHO
classification1 and to evaluate the presence of the following parameters:
angiocentrism/angioinvasion, epitheliotropism, and necrosis as well as to
assess the predominant small/medium- or large-cell component. In addition, the apoptotic index was determined, based on counting the absolute
number of apoptotic cells in 20 high-power fields (hpf’s). We used the
threshold of 10 apoptotic cells/hpf for statistical analysis. Detection of
apoptotic cells was made at distance from necrotic areas, using the
morphologic criteria as described by Walker et al.38
Immunohistochemical staining
Immunohistochemistry was performed on deparaffinized tissue sections
using an indirect immunoperoxidase method to evaluate T-, NK- and B-cell
differentiation antigens. After appropriate antigen retrieval, slides were
stained for CD20, CD3⑀ (DakoCytomation, Glostrup, Denmark), CD5,
GrB, CD56 (Novocastra, Newcastle, United Kingdom), and T-cell intracellular antigen 1 (TiA1; Immunotech, Marseille, France).
In addition, the expression of apoptotic-related proteins was investigated using antibodies against Bcl-2 (clone 124, DakoCytomation), the GrB
inhibitor PI9,39 whereas for aC3, the monoclonal rabbit antibody against
aC3 (PharMingen, San Diego, CA) was used. The 3 antibodies required
antigen retrieval by microwave irradiation in a citrate buffer (pH 6), after
which sections were incubated, respectively, overnight at 4°C (aC3) or for 1
hour at room temperature (PI9, Bcl-2). For aC3, a tyramide signal
amplification system (CSAII kit, DakoCytomation) was applied.
EBV studies
LMP-1 (CS1-4, DakoCytomation) was detected by immunohistochemistry.
The in situ hybridization (ISH) procedure for the detection of the
EBV-encoded RNA (EBER) transcripts was performed using the fluoresceinconjugated EBV (EBERs 1 and 2) oligonucleotides probes (DakoCytomation), as previously reported.40
Clinical staging and treatment
Performance status was based on the Eastern Cooperative Oncology Group
(ECOG) scale (0-4). Serum lactate dehydrogenase (LDH) level was
expressed as the ratio over the maximum normal value. Patients were
scored according to the International Prognostic Index (IPI).24
All patients but 3 were treated according to the consecutive LNH87,
LNH93, and LNH98 trials of the GELA, as previously published.26-35
Consequently, 42 patients received an anthracycline-based chemotherapy,
as follows: cyclophosphamide, hydroxydaunomycin, Oncovin, prednisone
(CHOP) regimen alone in 5 cases25,26 or followed by involved field
irradiation in 4 cases27; Adriamycin, cyclophosphamide, vindesine, bleomycin, prednisone (ACVBP) regimen, a dose-intensified CHOP-like combination, followed by consolidative sequential chemotherapy in 24 cases27-31; or
high-dose therapy with autologous transplantation in 329,31; cyclophosphamide, Oncovin, prednisone, Adriamycin/Cytoxan, vincristine, etoposide
(COPADM/CYVE) regimen in 6 patients.32 Six patients received non–
anthracycline-based regimens as follows: cyclophosphamide, vincristine,
prednisone (CVP) in 2 patients33; etoposide, Solu-Medrol, high-dose ara-C,
Platinol (ESHAP) in 2 patients34; and dexamethasone-gemcitabine, oxaliplatin (Dexa-GEMOX; gemcitabine 1000 mg/m2 intravenously on day 1,
oxaliplatin 100 mg/m2 intravenously on day 1, dexamethasone 40 mg daily,
given orally on day 1-4) in the last 2 patients.
Evaluation of tumor cells expressing Bcl-2, PI9, and aC3
A semiquantitative evaluation of PI9 and Bcl-2 expression was performed
by estimating the percentage of positive tumor cells and scored as follows:
⫺, 0% to 10%; ⫹, 10% to 25%; ⫹⫹, 25% to 50%; ⫹⫹⫹, 50% to 100%.
For the purpose of the study, a cut-off point of 10% stained tumor cells was
chosen for statistical analysis. Therefore cases with more than 10% PI9⫹
cells were scored as positive for PI9 expression and cases with less than
10% PI9⫹ cells as negative.
For aC3, 20 hpf’s in areas distant from necrosis were screened and the
mean value of positive tumor cells per field was calculated. Only malignant
cells with nuclear or nuclear/cytoplasmic staining were scored, thus
excluding macrophages with apoptotic debris. A cut-off of 10 aC3⫹ tumor
cells/hpf was chosen for statistical analysis.
Statistical analysis
Overall survival (OS) was measured from the date of diagnosis to the date
of death from any cause or last follow-up. Event-free survival (EFS) was
calculated from the date of diagnosis to disease progression, relapse, death,
or last follow-up. Survival curves were estimated using the method of
Kaplan-Meier41 and compared using the log-rank test.42 The prognostic
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BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
NASAL NK/T-CELL LYMPHOMA, PI9, AND OUTCOME
value for EFS and OS of the following parameters was studied by univariate
analysis: IPI score, histology, apoptotic index, and expression of PI9, aC3,
and Bcl-2. Multivariate analysis was performed using the Cox proportional
hazard model.43 Differences were considered significant when the 2-sided
P ⬍ .05.
All statistical analyses were performed using SAS 9.13 software (SAS
Institute, Cary, NC).
2185
Table 2. Histopathologic and phenotypic characteristics of the 48
patients with nasal NK/T-cell lymphomas
Characteristic
No. patients/total
(%)
Angiocentric/angioinvasion pattern
39/48 (81)
Epitheliotropism
37/48 (77)
Necrosis
42/48 (87)
Cytology
Results
Small/medium
22/48 (46)
Medium/large
26/48 (54)
CD3␧
Clinical characteristics of NK/T-cell lymphomas at presentation
The main clinical features are summarized in Table 1. There were
29 men and 19 women with a median age of 46 years. Most patients
(79%) presented with a localized stage I/II nasopharyngeal disease.
Ten patients had stage IV disease, as a result of involvement of
distant lymph nodes (n ⫽ 3), central nervous system (n ⫽ 2), or
bone marrow, lung, pleural cavity, liver, or skin (1 patient each).
The serum LDH level at diagnosis was normal in 34 (71%) patients
and the ECOG score was 0 to1 for 38 patients (79%). Thirty-one
patients (65%) had an IPI score less than 2.
Pathologic findings
The histopathologic findings are summarized in Table 2. Necrotic
areas were present in most cases (42 of 48, 87%), associated with
an angiocentric or angioinvasive growth pattern (or both) in 39 of
48 cases (81%). Variable degrees of epitheliotropism, defined by
the presence of clusters of CD3⑀⫹ tumor cells within the respiratory
glands or surface epithelia, were observed in 37 of 48 cases (77%).
NK/T-cell lymphomas were subclassified into 2 groups, according
to their predominant small- to medium-sized (22 cases, 46%) and
48/48 (100)
CD5
0/48 (0)
CD56
38/48 (79)
TiA1
32/32 (100)
GrB
35/35 (100)
EBV*
48/48 (100)
*EBV association was demonstrated by in situ hybridization in 47 cases and by
LMP-1 immunostaining in 6 of 10 cases.
large-sized (26 cases, 54%) cell features (Figure 1A-B). Small/
medium lymphoid cells disclosed a slightly irregular nucleus,
sometimes responsible for a centrocyte-like appearance, whereas
the large-cell category was characterized by a predominance of
cells with a large irregular nucleus, showing in a few cases
anaplastic features. In both groups, tumor cells frequently had an
Table 1. Clinical features and outcome of the 48 patients with nasal
NK/T-cell lymphomas and of the subgroup of 39 patients
evaluable for expression of PI9
Clinical features
No. of patients
No. male/no. female
Median age, y (range)
Entire group
Subgroup
48
39
29/19
27/12
46 (22-79)
46 (25-72)
Initial sites, no. (%)
Nasal cavity
32 (67)
27 (69)
Nasal cavity with adjacent structures
16 (33)
12 (31)
I
36 (75)
31 (80)
II
2 (4)
1 (2)
III
0 (0)
0 (0)
IV
10 (21)
7 (18)
0
25 (52)
23 (59)
1
13 (27)
8 (21)
2
2 (4)
2 (5)
3
8 (17)
6 (15)
Ann Arbor stage, no. (%)
ECOG score, no. (%)
LDH level, no. (%)
Normal
34 (71)
29 (74)
Elevated
14 (29)
10 (26)
IPI score, no. (%)
0
22 (46)
18 (47)
1
9 (19)
8 (20)
2
9 (19)
8 (20)
3
5 (10)
3 (8)
4
3 (6)
2 (5)
5-y EFS rate
39
41
5-y OS rate
49
50
Survival, %
Figure 1. Histologic subtypes of nasal NK/T-cell lymphomas and detection of
PI9 and aC3 in tumors cells of nasal NK/T-cell lymphomas. (A) Example of a
lymphoma with a predominant small to medium-sized cell component with mild
atypia. (B) Example of a lymphoma with pleomorphic large-cell features. (C) Example
of a nasal NK/T-cell lymphoma scored as positive for PI9, with virtually all tumor cells
surrounding glands positive. (D) Another nasal NK/T-cell lymphoma scored as
negative for PI9 (⬍ 10% tumor cells positive). A few scattered cells serve as internal
positive control. (E) Example of a nasal NK/T-cell lymphoma with more than 10%
aC3⫹ tumor cells (brown nuclear staining), whereas panel F illustrates a case with
less than 10% aC3⫹ tumor cells. Only rare scattered cells with brown nuclear staining
are present. Original magnification, ⫻ 400 for panels A-B, ⫻ 100 for panel C, and
⫻ 200 for panels D-F). Images were captured with a Zeiss Axioskop2 microscope
(Oberkochen, Germany) and Neofluar 100⫻/0.1 NA optical lenses (Zeiss). Photographs were taken with a DP70 Olympus camera (Tokyo, Japan). Image acquisition
was performed with Olympus DP Controller 2002, and images were processed with
Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA).
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BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
BOSSARD et al
abundant clear cytoplasm. The apoptotic index was variable from
case to case and was scored as high (⬎ 10 apoptotic tumor
cells/hpf) in 12 of 37 (32%) evaluable cases. Interestingly, it was
higher in the large cell category than in the small/medium-cell
category (32 and 4 apoptotic cells/hpf, respectively, P ⫽ .009). On
immunohistochemistry, all lymphomas disclosed a characteristic
CD3⑀⫹/CD5⫺ phenotype with expression of GrB or TiA1 cytotoxic
molecules, whereas expression of CD56 was detected in 38 of 48
cases (79%). EBV association was demonstrated in all cases either
by EBER ISH (n ⫽ 47 of 48) or LMP-1 immunostaining (n ⫽ 6 of 10).
Expression of PI9, aC3, and Bcl-2 proteins
in NK/T-cell lymphomas
Due to insufficient tissue or inadequate staining, a few cases could
not be evaluated for expression of PI9, aC3, and Bcl-2 (Table 3). To
note, as shown in Table 1, the subgroup of 39 patients evaluable for
PI9 did not differ for the distribution of clinical characteristics. In
agreement with the known constitutive expression of GrB and PI9
by normal NK cells,21 all evaluable NK/T-cell lymphomas disclosed positivity for both markers. However, the expression level
of PI9 was heterogeneous from case to case and PI9 was scored as
positive (eg, ⬎ 10% tumor cells) in 26 of 39 (67%) evaluable cases
and as negative in 13 of 39 (33%) cases (Figure 1C-D). In PI9⫹
cases, the immunostaining was usually strong in virtually all tumor
cells, but in a few cases it was heterogeneous, with positive clusters
of neoplastic cells in a negative background.
Regarding aC3, patients were distributed into low (n ⫽ 23) and
high (n ⫽ 13) expression groups (Figure 1E-F). Although most
aC3⫹ tumor cells disclosed morphologic features of apoptosis, the
number of aC3-labeled cells was overall smaller than that of
morphologic apoptotic cells. Furthermore, no correlation was
found between the level of PI9 and aC3. Finally, expression of
Bcl-2 was scored as positive in at least 10% of tumor cells in only 6
of 25 (24%) tested cases.
Clinical outcome and prognostic parameters
CR/CRu to first-line treatment was achieved in 24 of 48 patients
(50%), whereas 21 patients progressed and 3 died from disease
during treatment. Five patients had a relapse, most often locally (4
cases). The median time to relapse was 20 months (range, 2-54
months), with 3 patients having the relapse in the first 2 months.
Patients in failure or in relapse received a salvage treatment
consisting of exclusive chemotherapy in 15 cases and chemotherapy followed by involved-field radiotherapy in 11 cases.
Among the 29 deaths registered, 27 resulted from lymphoma
progression and 2 occurred in patients with persistent CR (myocardial infarction and radiotherapy-induced myelitis). With a median
follow-up of 6.5 years, the 5-year estimates for EFS and OS were
39% and 49%, respectively (Figure 2). As shown in Table 1, the
subgroup of 39 patients evaluable for PI9 did not differ for 5-year
EFS and OS rates (41% and 50%, respectively).
By univariate analysis (Table 3), expression of PI9 by tumor
cells was associated with a highly significant better 5-year OS
(P ⫽ .009) and 5-year EFS (P ⫽ .02), as compared with PI9⫺
lymphomas (Figure 3A-B). A high apoptotic index (Figure 4), but
not expression of aC3, was also associated with a significantly
better 5-year OS (P ⫽ .05) and a trend for a better 5-year EFS
(P ⫽ .08). A low IPI score (⬍ 2) was associated with a trend for a
better 5-year OS (P ⫽ .06). In a multivariate analysis (Table 4), OS
and EFS were independently affected by PI9 expression (P ⫽ .009
and P ⫽ .02, respectively) and apoptotic index (P ⫽ .05 and
P ⫽ .08, respectively).
Discussion
We report here a series of 48 nasal NK/T-cell lymphomas, the
largest so far reported Occidental series,5,44-47 in which diagnosis
was based on strict histopathologic and phenotypic criteria including the demonstration of both a cytotoxic profile and EBV
association. Following first-line chemotherapy with curative intent
in most patients, we confirm the poor prognosis of the disease, as
already reported with other treatment modalities.4,6,9 Interestingly,
we show that the level of the GrB inhibitor PI9 and the apoptotic
index are independent prognostic indicators.
In keeping with previous Asian and Western reports,2,6,8-10,12,43,44,46-51
we observed that the disease has a male predominance with a median
age around 50 years and presents as a localized disease restricted to the
nasal cavity and adjacent structures. As others,6,9,48,49 we found few
adverse risk factors in the majority of the patients, resulting in a low
(⬍ 2) IPI score. We also confirm that the disease pursues a highly
aggressive clinical course with only half the patients achieving a CR to
Table 3. Univariate analysis of prognostic factors in patients with nasal NK/T-cell lymphoma
Prognostic factor
No. of
patients
Estimated OS
5-y rate, %
Expression of P19*
P
Estimated EFS
5-y rate, %
.009
Positive, more than 10% of tumor cells
26
64 ⫾ 9†
61 ⫾ 10†
Negative 10% or less of tumor cells
13
23 ⫾ 11†
23 ⫾ 12†
Low, 10 or fewer cells/hpf
25
36 ⫾ 10†
High, more than 10 cells/hpf
12
67 ⫾ 14†
Apoptotic index*
.05
IPI score
.08
32 ⫾ 10†
58 ⫾ 14†
.06
Lower than 2
31
59 ⫾ 9†
Higher than 2
17
29 ⫾ 11†
Histology
.2
47
29
.4
Small/medium cells
22
43
Medium/large cells
26
53
Expression of aC3*
P
.02
.3
39
49
.8
.7
Low, fewer than 10 tumor cells
23
43
39
High, 10 or more tumor cells
13
46
38
*In 11 cases, the small size of the sections or abundant areas of necrosis did not allow the assessment of the number of apoptotic cells on 20 hpf’s. Due to insufficient tissue
or inadequate staining, a few cases could not be evaluated for expression of PI9 (n ⫽ 9) and aC3 (n ⫽ 13).
†95% CI.
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BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
NASAL NK/T-CELL LYMPHOMA, PI9, AND OUTCOME
2187
Figure 4. OS according apoptotic index. P ⫽ .05
Figure 2. Survival statistics. (A) EFS for all patients. (B) OS for all patients.
treatment and surviving at 5 years. The optimal therapy of nasal
NK/T-cell lymphoma remains debated because trials comparing prospectively radiotherapy with chemotherapy are lacking. However, several
studies have suggested radiotherapy-based primary treatment to be
superior to chemotherapy alone.3-5,7,9,11 In our series, all patients but 4
received chemotherapy alone as primary treatment. This approach
resulted in a 49% 5-year survival rate, which compares to that reported
in patients receiving radiotherapy-based primary treatment.3,4,6,7,11,48,49
Figure 3. Survival according to PI9 expression. (A) EFS, P ⫽ .02. (B) OS,
P ⫽ .009.
Moreover, in our study, salvage with combined chemoradiotherapy
appeared to be ineffective with only 1 of 11 cases in persistent second
remission (data not shown).
Several attempts have been made to identify prognostic parameters in nasal NK/T-cell lymphomas. Advanced stage,4,9,11,50-52 high
IPI score,7-9,51-53 and local tumor invasiveness10 have been reported
to predict an unfavorable outcome. Recently, Lee et al48 proposed a
prognostic model based on B symptoms, stage, LDH level, and
regional lymph node involvement allowing a better prognostic
discrimination as compared with IPI. There have been conflicting
results regarding the relevance of cytologic grading in nasal
NK/T-cell lymphomas53,54; our results in patients receiving primary
chemotherapy do not support its prognostic impact.
Efforts are also ongoing in nasal NK/T-cell lymphoma to
identify molecular abnormalities with clinical relevance that may
help in defining mechanisms responsible for resistance to therapyinduced apoptosis and ultimately alternative therapies. For instance, P53 missense mutations have been associated with an
aggressive course,54 whereas overexpression of CD94, an NK-cell
antigen receptor, has been shown to predict a favorable outcome.55
In the present study, we show for the first time that high level of
expression of the putative antiapoptotic protein PI9 by lymphoma
cells is, unexpectedly, associated with a favorable outcome. This is
in contrast with a previous report in anaplastic large-cell lymphoma23 showing that PI9 expression by neoplastic cells was
associated with a poor outcome, an observation in keeping with the
hypothesis that tumor cells use expression of PI9 to escape their
elimination mediated by the release of GrB by cytotoxic cells.22
These discrepancies may be explained by the constitutive expression of both GrB and PI9 by NK cells,56 to protect themselves from
a premature cell death. Indeed, GrB and PI9 form complexes into
the cytoplasm of NK cells immediately after leakage of GrB from
intracellular granules to rapidly inactivate extra granular GrB.57 On
the other hand, the assumption that PI9 may confer resistance to
apoptosis was not confirmed in a recent report.58 In this study,
human lymphoma cell lines and primary lymphoma cells were
sensitive, independently of their level of PI9 expression, to
cytolysis by both CTLs and activated NK cells. Furthermore,
recent data suggest that PI9 may become inactivated through its
cleavage by granzyme M,59 another enzyme present in cytotoxic
granules, which is specifically expressed by cells of the innate
immune system and also by nasal NK/T-cell lymphomas.60 Finally,
PI9 belongs to the serpin superfamily, which comprises several
members constitutively expressed in epithelial cells.61,62 The expression of serpins has been shown to be down-regulated in several
invasive carcinomas63,64 and the corresponding genes have been
regarded as tumor suppressor genes, able to suppress tumor
growth, invasion, and metastasis and to enhance tumor cell
sensitivity to apoptosis. Interestingly, tumor cells expressing one of
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2188
BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
BOSSARD et al
Table 4. Multivariate analysis of prognostic factors in patients with
nasal NK/T-cell lymphoma
OS
EFS
Prognostic
factor
P
Risk
ratio
95% CI
P
Risk
ratio
95% CI
Expression of PI9
.009
3.55
2.19-5.88
.02
2.98
1.86-4.76
Apoptotic index
.04
3.75
2.87-4.9
.03
4
2.09-7.62
IPI score
.1
0.45
.16
0.53
these serpins, named maspin, have a reduced level of bcl-2
protein,65 a finding that parallels our observation of a lack of Bcl-2
in most cases of nasal NK/T-cell lymphomas in the present series.
In the light of these data in epithelial tumors, it is tempting to
speculate that the loss of PI9 in nasal NK/T-cell lymphomas could
reflect a dedifferentiation process associated with tumor progression, resistance to apoptosis, and ultimately poor outcome. Interestingly, we observed in some PI9⫹ cases an heterogeneous PI9
immunostaining with clusters of negative cells, which might
suggest the emergence of PI9 down-regulated subclones possibly
associated with aggressiveness and invasive potential. The mechanism by which NK/T tumor cells may down-regulate PI9 needs to
be clarified. Cytogenetic studies have shown 6q deletion as a
consistent aberration in NK/T-cell lymphomas,66 whereas PI9 is
located at chromosome 6p.67 It is tempting to speculate that PI9
down-regulation might result from epigenetic inactivations by
aberrant promoter hypermethylation, as described for tumor suppressor genes such as p73 in NK-cell disorders.68
In contrast to the association of a high apoptotic index with a
favorable outcome, we observed a broad range of aC3 expression
without correlation with prognosis. In most cases, the number of
apoptotic cells appeared higher than the number of aC3⫹ cells,
resulting in the absence of correlation between both parameters.
Although the reasons for such discrepancies are unclear, it might
reflect the involvement of a caspase-independent programmed cell
death, as recently reported.69
We conclude that further studies at a large multicenter scale,
with respect to the rarity of the disease, are warranted to confirm
the prognostic relevance of PI9 expression in nasal NK/T-cell
lymphomas. Such studies are facilitated by the fact that PI9
expression can be easily evaluated on routinely fixed material.
They should evaluate whether PI9 expression, in addition to other
clinical prognostic factors such as local tumor invasiveness and IPI,
could help to stratify patients with nasal NK/T-cell lymphomas and
possibly propose adapted innovative therapies.
Acknowledgment
We indebted to Chafika Coppeaux and Karine Sardin for their
technical help and to Nathalie Dordonne for her help in statistical analysis.
Authorship
C.B., K.B., F.R. and P.G. wrote the paper; N.M.-G. and A.K.
performed the experiments and provided the PI9 monoclonal
antibody; C.B., P.G., F.B., J.B., A.C.-B., and J.B. participated in the
pathologic review of patients entered in the GELA studies; K.B.,
P.G., N.M., and F.R. analyzed the data; F.R., V.R., C.G., and N.M.
participated in designing the LNH87, LNH93, LNH98 studies and
treating and documenting the patients.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Felix Reyes died on August 13, 2006.
A list of the participating members of the GELA is presented in
Document S1 (available at the Blood website; see the Supplemental
Document link at the top of the online version of the article).
Correspondence: Philippe Gaulard, Département de pathologie,
Hôpital Henri Mondor, 51, Avenue du Maréchal de Lattre de Tassigny,
94010 Créteil Cedex, France; e-mail: [email protected].
References
1. Jaffe ES, Lee Harris N, Stein H, Vardiman JW.
Tumors of haematopoietic and lymphoid tissues.
World Health Organization Classification of Tumors. Lyon, France: IARC Press; 2001.
8. Chim CS, Ma SY, Au WY, et al. Primary nasal
natural killer cell lymphoma: long-term treatment
outcome and relationship with the International
Prognostic Index. Blood. 2004;103:216-221.
2. Arnulf B, Copie-Bergman C, Delfau-Larue MH, et
al. Nonhepatosplenic gammadelta T-cell lymphoma: a subset of cytotoxic lymphomas with
mucosal or skin localization. Blood. 1998;91:
1723-1731.
9. You JY, Chi KH, Yang MH, et al. Radiation
therapy versus chemotherapy as initial treatment
for localized nasal natural killer (NK)/T-cell lymphoma: a single institute survey in Taiwan. Ann
Oncol. 2004;15:618-625.
3. Aviles A, Diaz NR, Neri N, Cleto S, Talavera A.
Angiocentric nasal T/natural killer cell lymphoma:
a single centre study of prognostic factors in 108
patients. Clin Lab Haematol. 2000;22:215-220.
10. Kim TM, Park YH, Lee SY, et al. Local tumor invasiveness is more predictive of survival than International Prognostic Index in stage I(E)/II(E) extranodal NK/T-cell lymphoma, nasal type. Blood.
2005;106:3785-3790.
4. Kim GE, Cho JH, Yang WI, et al. Angiocentric
lymphoma of the head and neck: patterns of systemic failure after radiation treatment. J Clin Oncol. 2000;18:54-63.
11. Li YX, Yao B, Jin J, et al. Radiotherapy as primary
treatment for stage IE and IIE nasal natural killer/
T-cell lymphoma. J Clin Oncol. 2006;24:181-189.
Granzyme B: a natural born killer. Immunol Rev.
2003;193:31-38.
16. Rathmell JC, Thompson CB. The central effectors
of cell death in the immune system. Annu Rev
Immunol. 1999;17:781-828.
17. Dukers DF, Meijer CJ, ten Berge RL, Vos W, Ossenkoppele GJ, Oudejans JJ. High numbers of
active caspase 3-positive Reed-Sternberg cells in
pretreatment biopsy specimens of patients with
Hodgkin disease predict favorable clinical outcome. Blood. 2002;100:36-42.
18. Green DR, Kroemer G. The pathophysiology of
mitochondrial cell death. Science. 2004;305:626629.
19. Hermine O, Haioun C, Lepage E, et al. Prognostic significance of bcl-2 protein expression in aggressive non-Hodgkin’s lymphoma. Groupe
d’Etude des Lymphomes de l’Adulte (GELA).
Blood. 1996;87:265-272.
5. Ribrag V, Ell Hajj M, Janot F, et al. Early locoregional high-dose radiotherapy is associated with
long-term disease control in localized primary angiocentric lymphoma of the nose and nasopharynx. Leukemia. 2001;15:1123-1126.
12. Los M, Herr I, Friesen C, Fulda S, Schulze-Osthoff K, Debatin KM. Cross-resistance of CD95and drug-induced apoptosis as a consequence of
deficient activation of caspases (ICE/Ced-3 proteases). Blood. 1997;90:3118-3129.
6. Cheung MM, Chan JK, Lau WH, Ngan RK, Foo
WW. Early stage nasal NK/T-cell lymphoma: clinical outcome, prognostic factors, and the effect of
treatment modality. Int J Radiat Oncol Biol Phys.
2002;54:182-190.
13. Wesselborg S, Engels IH, Rossmann E, Los M,
Schulze-Osthoff K. Anticancer drugs induce
caspase-8/FLICE activation and apoptosis in the
absence of CD95 receptor/ligand interaction.
Blood. 1999;93:3053-3063.
7. Li CC, Tien HF, Tang JL, et al. Treatment outcome
and pattern of failure in 77 patients with sinonasal
natural killer/T-cell or T-cell lymphoma. Cancer.
2004;100:366-375.
14. Creagh EM, Conroy H, Martin SJ. Caspase-activation pathways in apoptosis and immunity. Immunol Rev. 2003;193:10-21.
21. Bird CH, Sutton VR, Sun J, et al. Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing
the Fas cell death pathway. Mol Cell Biol. 1998;
18:6387-6398
15. Lord SJ, Rajotte RV, Korbutt GS, Bleackley RC.
22. Bladergroen BA, Meijer CJ, ten Berge RL, et al.
20. Mounier N, Briere J, Gisselbrecht C, et al. Rituximab plus CHOP (R-CHOP) overcomes bcl-2–
associated resistance to chemotherapy in elderly
patients with diffuse large B-cell lymphoma (DLBCL). Blood. 2003;101:4279-4284.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 MARCH 2007 䡠 VOLUME 109, NUMBER 5
Expression of the granzyme B inhibitor, protease
inhibitor 9, by tumor cells in patients with nonHodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the
immune system? Blood. 2002;99:232-237
23. ten Berge RL, Meijer CJ, Dukers DF, et al. Expression levels of apoptosis-related proteins predict clinical outcome in anaplastic large cell lymphoma. Blood. 2002;99:4540-4546.
24. A predictive model for aggressive non-Hodgkin’s
lymphoma. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. N Engl J Med.
1993;329:987-994.
25. Tilly H, Lepage E, Coiffier B, et al. Intensive conventional chemotherapy (ACVBP regimen) compared with standard CHOP for poor-prognosis
aggressive non-Hodgkin lymphoma. Blood. 2003;
102:4284-4289.
26. Fillet G, Bonnet C, Mounier N, et al. No role for
chemoradiotherapy when compared with chemotherapy alone in elderly patients with localized
low risk aggressive lymphoma: final results of the
LNH93–4 GELA study [abstract]. Blood. 2005;
106:15a. Abstract 15.
27. Reyes F, Lepage E, Ganem G, et al.ACVBP versus CHOP plus radiotherapy for localized aggressive lymphoma. N Engl J Med. 2005;352:11971205.
28. Tilly H, Mounier N, Lederlin P, et al. Randomized
comparison of ACVBP and m-BACOD in the
treatment of patients with low-risk aggressive
lymphoma: the LNH87–1 study. Groupe d’Etudes
des Lymphomes de l’Adulte. J Clin Oncol. 2000;
18:1309-1315.
29. Haioun C, Lepage E, Gisselbrecht C, et al. Survival benefit of high-dose therapy in poor-risk aggressive non-Hodgkin’s lymphoma: final analysis
of the prospective LNH87–2 protocol—a groupe
d’Etude des lymphomes de l’Adulte study. J Clin
Oncol. 2000;18:3025-3030.
30. Bosly A, Lepage E, Coiffier B, et al. Outcome is
not improved by the use of alternating chemotherapy in elderly patients with aggressive lymphoma. Hematol J. 2001;2:279-285.
31. Gisselbrecht C, Lepage E, Molina T, et al. Shortened first-line high-dose chemotherapy for patients with poor-prognosis aggressive lymphoma.
J Clin Oncol. 2002;20:2472-2479.
32. Delmer A, Mounier N, Gaulard P, et al. Intensified
induction therapy with etoposide (VP16) and
high-dose cytarabine (Ara-C) in patients aged
less than 60 years with peripheral T cell/NK lymphoma: preliminary results of the phase II GELA
study LNH98T7 [abstract]. Proc Am Soc Clin Oncol. 2003;22:2375a.
33. Bastion Y, Blay JY, Divine M, et al. Elderly patients with aggressive non-Hodgkin’s lymphoma:
disease presentation, response to treatment, and
survival—a Groupe d’Etude des Lymphomes de
l’Adulte study on 453 patients older than 69
years. J Clin Oncol. 1997;15:2945-2953.
34. Bouabdallah R, Delmer A, Xerri L, Mounier N,
Reyes F. ESHAP chemotherapy regimen and 13cis-retinoic acid in elderly patients with untreated
poor-prognosis peripheral T-cell lymphoma: a
GELA phase II trial of feasibility and efficacy [abstract]. Ann Oncol. 2005;16(suppl 5):322a. Abstract 322.
35. Cheson BD, Horning SJ, Coiffier B, et al. Report
of an international workshop to standardize response criteria for non-Hodgkin’s lymphomas.
NCI Sponsored International Working Group.
J Clin Oncol. 1999;17:1244-1253.
36. Stansfeld AG, Diebold J, Noel H, et al. Updated
NASAL NK/T-CELL LYMPHOMA, PI9, AND OUTCOME
Kiel classification for lymphomas. Lancet. 1988;1:
292-293.
37. National Cancer Institute sponsored study of
classifications of non-Hodgkin’s lymphomas:
summary and description of a working formulation for clinical usage. The Non-Hodgkin’s Lymphoma Pathologic Classification Project. Cancer.
1982;49:2112-2135.
38. Walker NI, Harmon BV, Gobe GC, Kerr JF. Patterns of cell death. Methods Achiev Exp Pathol.
1988;13:18-54.
39. Bladergroen BA, Strik MC, Bovenschen N, et al.
The granzyme B inhibitor, protease inhibitor 9, is
mainly expressed by dendritic cells and at immune-privileged sites. J Immunol. 2001;166:
3218-3225.
40. Kanavaros P, Lescs MC, Briere J, et al. Nasal Tcell lymphoma: a clinicopathologic entity associated with peculiar phenotype and with EpsteinBarr virus. Blood. 1993;81:2688-2695.
41. Kaplan EL, Meier, P. Non parametric estimation
from incomplete observations. J Am Stat Assoc.
1958:457-481.
42. Mantel N. Evaluation of survival data and two
new rank order statistics arising in its consideration. Cancer Chemother Rep. 1966;50:163-170.
43. Cox DR. Regression models and life-tables (with
discussion). 1972;34:187-195.
44. Campo E, Cardesa A, Alos L, et al. NonHodgkin’s lymphomas of nasal cavity and paranasal sinuses. An immunohistochemical study.
Am J Clin Pathol. 1991;96:184-190.
45. Ferry JA, Sklar J, Zukerberg LR, Harris NL. Nasal
lymphoma. A clinicopathologic study with immunophenotypic and genotypic analysis. Am J Surg
Pathol. 1991;15:268-279.
46. Proulx GM, Caudra-Garcia I, Ferry J, et al. Lymphoma of the nasal cavity and paranasal sinuses:
treatment and outcome of early-stage disease.
Am J Clin Oncol. 2003;26:6-11.
47. Pagano L, Gallamini A, Trape G, et al. NK/T-cell
lymphomas ‘nasal type’: an Italian multicentric
retrospective survey. Ann Oncol. 2006;17:794800.
48. Lee J, Suh C, Park YH et al. Extranodal natural
killer T-cell lymphoma, nasal-type: a prognostic
model from a retrospective multicenter study.
J Clin Oncol. 2006;24:612-618.
49. Kim K, Chie EK, Kim CW, Kim IH, Park CI. Treatment outcome of angiocentric T-cell and NK/Tcell lymphoma, nasal type: radiotherapy versus
chemoradiotherapy. Jpn J Clin Oncol. 2005;35:
1-5.
50. Rudiger T, Weisenburger DD, Anderson JR, et al.
Peripheral T-cell lymphoma (excluding anaplastic
large-cell lymphoma): results from the NonHodgkin’s Lymphoma Classification Project. Ann
Oncol. 2002;13:140-149.
51. Kim BS, Kim TY, Kim CW, et al. Therapeutic outcome of extranodal NK/T-cell lymphoma initially
treated with chemotherapy—result of chemotherapy in NK/T-cell lymphoma. Acta Oncol.
2003;42:779-783.
52. Lee J, Kim WS, Park YH, et al. Nasal-type NK/T
cell lymphoma: clinical features and treatment
outcome. Br J Cancer. 2005;92:1226-1230.
53. Ng SB, Lai KW, Murugaya S, et al. Nasal-type
extranodal natural killer/T-cell lymphomas: a clinicopathologic and genotypic study of 42 cases in
Singapore. Mod Pathol. 2004;17:1097-1107.
54. Takahara M, Kishibe K, Bandoh N, Nonaka S,
Harabuchi Y. P53, N- and K-Ras, and beta-catenin gene mutations and prognostic factors in na-
2189
sal NK/T-cell lymphoma from Hokkaido, Japan.
Hum Pathol. 2004;35:86-95.
55. Lin CW, Chen YH, Chuang YC, Liu TY, Hsu SM.
CD94 transcripts imply a better prognosis in nasal-type extranodal NK/T-cell lymphoma. Blood.
2003;102:2623-2631.
56. Hirst CE, Buzza MS, Bird CH, et al. The intracellular granzyme B inhibitor, proteinase inhibitor 9,
is up-regulated during accessory cell maturation
and effector cell degranulation, and its overexpression enhances CTL potency. J Immunol.
2003;170:805-815.
57. Ida H, Nakashima T, Kedersha NL, et al. Granzyme B leakage-induced cell death: a new type of
activation-induced natural killer cell death. Eur
J Immunol. 2003;33:3284-3292.
58. Godal R, Keilholz U, Uharek L, et al. Lymphomas
are sensitive to perforin-dependent cytotoxic
pathways despite expression of PI-9 and overexpression of bcl-2. Blood. 2006;107:3205-3211.
59. Mahrus S, Kisiel W, Craik CS. Granzyme M is a
regulatory protease that inactivates proteinase
inhibitor 9, an endogenous inhibitor of granzyme
B. J Biol Chem. 2004;279:54275-54282.
60. Krenacs L, Smyth MJ, Bagdi E, et al. The serine
protease granzyme M is preferentially expressed
in NK-cell, gamma delta T-cell, and intestinal Tcell lymphomas: evidence of origin from lymphocytes involved in innate immunity. Blood. 2003;
101:3590-3593.
61. Cataltepe S, Gornstein ER, Schick C, et al. Coexpression of the squamous cell carcinoma antigens 1 and 2 in normal adult human tissues and
squamous cell carcinomas. J Histochem Cytochem. 2000;48:113-122.
62. Futscher BW, O’Meara MM, Kim CJ, et al. Aberrant methylation of the maspin promoter is an
early event in human breast cancer. Neoplasia.
2004;6:380-389.
63. Lockett J, Yin S, Li X, Meng Y, Sheng S. Tumor
suppressive maspin and epithelial homeostasis.
J Cell Biochem. 2006;97:651-660.
64. Shellenberger TD, Mazumdar A, Henderson Y, et
al. Headpin: a serpin with endogenous and exogenous suppression of angiogenesis. Cancer Res.
2005;65:11501-11509.
65. Zhang W, Shi HY, Zhang M. Maspin overexpression modulates tumor cell apoptosis through the
regulation of Bcl-2 family proteins. BMC Cancer.
2005;5:50-61.
66. Wong KF, Chan JK, Kwong YL. Identification of
del(6)(q21q25) as a recurring chromosomal abnormality in putative NK cell lymphoma/leukaemia. Br J Haematol. 1997;98:922-926.
67. Eyre HJ, Sun J, Sutherland GR, Bird P. Chromosomal mapping of the gene (PI9) encoding the
intracellular serpin proteinase inhibitor 9 to 6p25
by fluorescence in situ hybridization. Genomics.
1996;37:406-408.
68. Kawamata N, Inagaki N, Mizumura S, et al. Methylation status analysis of cell cycle regulatory
genes (p16INK4A, p15INK4B, p21Waf1/Cip1,
p27Kip1 and p73) in natural killer cell disorders.
Eur J Haematol. 2005;74:424-429.
69. Abraham MC, Shaham S. Death without
caspases, caspases without death. Trends Cell
Biol. 2004;14:184-193.
70. Bossard C, Belhadj K, Reyes F, et al. Primary nasal NK/T cell lymphoma: cytology and level of the
antiapoptotic PI9 protein have prognostic relevance [abstract]. Blood. 2004;104:891a. Abstract 3264.
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2007 109: 2183-2189
doi:10.1182/blood-2006-07-033142 originally published
online October 31, 2006
Expression of the granzyme B inhibitor PI9 predicts outcome in nasal
NK/T-cell lymphoma: results of a Western series of 48 patients treated
with first-line polychemotherapy within the Groupe d'Etude des
Lymphomes de l'Adulte (GELA) trials
Céline Bossard, Karim Belhadj, Felix Reyes, Nadine Martin-Garcia, Françoise Berger, Jean Alain
Kummer, Josette Brière, Anne-Catherine Baglin, Stéphane Cheze, Jacques Bosq, Vincent Ribrag,
Christian Gisselbrecht, Nicolas Mounier and Philippe Gaulard
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