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Prepublished online as a Blood First Edition Paper on November 21, 2002; DOI 10.1182/blood-2002-07-2215.
NEOPLASIA
From the Département de Pathologie, the Service
d'Immunologie Biologique, and the Service d'Hématologie
Clinique, EA2348, AP-HP, Hôpital Henri Mondor, Créteil,
France; the Institute of Pathology, University of Ulm,
Germany; the Department of Pathology, University of
Leicester, United Kingdom; and the Département
d'Hématologie of Institut Cochin, Maternité Port-Royal,
Paris, France.
The molecular markers that distinguish primary mediastinal large
B-cell lymphoma (PMBL) from nonmediastinal diffuse large B-cell
lymphomas (NM-DLBLs) remain to be identified. Using cDNA representational difference analysis to compare PMBL and NM-DLBL transcripts, we isolated a cDNA fragment homologous to the
mouse B-cell interleukin 4 (IL-4)-inducible gene FIG1
(interleukin 4-induced gene 1) transcript. The human FIG1
mRNA encodes a 567 amino acid protein that comprises a signal peptide
and a large flavin-binding amino oxidase domain, and shares significant
homology with secreted apoptosis-inducing L-amino acid oxidases.
Northern blot studies showed that FIG1 mRNA expression is
mainly restricted to lymphoid tissues. It is expressed at low levels in
thymus, spleen, tonsils, and reactive lymph nodes, and is
highly up-regulated in IL-4+CD40-activated tonsillar B cells.
Interestingly, in human B-cell lines, FIG1 mRNA expression
appeared restricted to the PMBL-derived MedB-1 and Karpas 1106 cell
lines. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrated that all but one PMBL (16/17)
displayed high FIG1 mRNA levels, whereas most
NM-DLBLs (12/18) and all low-grade B-cell lymphomas tested (8/8)
exhibited low FIG1 mRNA levels. The difference between
PMBLs and NM-DLBLs was statistically significant (Fisher test;
P = .0003). Southern blot studies did not show
rearrangement of the FIG1 gene. FIG1 gene
expression might be due to a constitutive activation of a cytokine
signaling pathway in PMBL.
(Blood. 2003;101:2756-2761) In the past 10 years, there has been increasing
evidence that primary mediastinal large B-cell lymphoma (PMBL) harbors
unique clinical, pathologic, and immunohistochemical features. This
disease is now recognized as a specific lymphoma subtype among diffuse large B-cell lymphomas (DLBLs) in the World Health Organization (WHO)
classification.1 PMBL accounts for about 5% of aggressive lymphomas, tends to affect a relatively young population (average age
37 years), and presents a 50% to 60% 5-year failure-free survival rate despite intensive chemotherapy.2 It is characterized
by a rapidly growing mass, developing in the anterior upper
mediastinum, consisting of large B cells that usually express little if
any surface or cytoplasmic immunoglobulin and often lack major
histocompatibility complex (MHC) class I and/or class II
molecules.3-5 It is now assumed that PMBL derives from a
peculiar population of thymic medullary B cells.6,7
Attempts to assign PMBL precursor B cells to a specific developmental
stage have led to controversial views.8,9 However, recent
data based on immunoglobulin gene analysis suggest that PMBL is of
post-germinal center origin.10
Although the clinical and pathologic characteristics of PMBL are now
well recognized, a tumor-specific gene expression profile and specific
chromosomal translocations have not yet been reported. This may be
explained by the rarity of the disease and the difficulty to obtain
fresh material for molecular studies. BCL2 and
BCL6 rearrangements usually observed in DLBL appear to be
rare in PMBL.11,12 Comparative genomic hybridization
studies have reported frequent overrepresentations of genomic segments
of chromosome 9p, Xq, 12q, and 2p.13,14 Several candidate
genes have been proposed, among which are the proto-oncogene
c-REL and the Janus kinase 2 (JAK2)
gene,15 but still, none of them has been specifically assigned to PMBL lymphomagenesis.
Hence, more accurate PMBL gene expression profiling appears necessary
not only to improve diagnosis and treatment, but also to understand the
molecular alterations involved in the pathogenesis of this particular
lymphoma subtype. In a recent study, we used differential display
reverse transcription to compare the mRNAs expressed in PMBL with those
expressed in nonmediastinal DLBLs (NM-DLBLs).16 We
identified the MAL gene as a distinct molecular marker of
PMBL. The MAL gene is normally expressed in lymphoid T
cells, polarized epithelial cells, and myelin-forming
cells.17-19 It encodes a proteolipid believed to
participate in membrane microdomains stabilization, intracellular
transport, and signaling.20,21 Its expression in PMBL may
modify raft dynamics and contribute to neoplastic transformation.
To extend PMBL gene expression profile analysis, we used
representational difference analysis (RDA) to isolate genes that are
differentially expressed in PMBL versus NM-DLBL. In this report, we
describe the identification of a gene that is frequently activated in
PMBL and is the human homologue of the mouse immediate-early interleukin 4 (IL-4)-inducible gene FIG1 (interleukin
4-induced gene 1).22
Tissue specimens and cell lines
Normal and reactive lymphoid tissues were used as a control. These
included one tonsil from a child with follicular hyperplasia; 3 lymph
node biopsies showing benign follicular hyperplasia; one spleen
obtained from a patient with autoimmune disorder; and one thymus
removed at necropsy from a 40-week-gestation fetus. All specimens were
received fresh, and samples were snap frozen or processed immediately.
Highly purified B cells were obtained from tonsillar cell
suspensions by positive selection using CD19 magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells were activated for 6 days in 25 cm2 culture flasks with irradiated (70 Gy)
transfected murine L cells expressing human CD40 ligand and 10 ng/mL
IL-4 (Sanofi, Labège, France). Activated B cells were more than
98% CD19+, CD80+, and CD86+.
B-cell lines used in this study included the B-cell precursor
acute lymphoblastic leukemia (ALL) cell lines RS 4:11, Nalm6, Nalm16, and 697; the Burkitt lymphoma cell line Ramos; the RL lymphoma
cell line bearing a t(14;18) translocation; and 2 cell lines derived
from patients with PMBL in relapse, Karpas 1106 and
MedB-1.23,24 Other hematopoietic (Jurkat, SUDHL1, Peer and
DU528 T-cell lines, HEL erythro-megakaryocytic cell line) and
nonhematopoietic (HepG2 hepatoma cell line, MZ2 melanoma cell line, and
Hela epithelial cell line) cell lines were also studied.
Representational difference analysis subtraction and screening
Northern blot analysis Total RNA was isolated using TRIzol reagent. Total RNA (5 µg) was fractionated in a 1% agarose gel containing formaldehyde and transferred to Hybond-N+ membranes (Amersham Biosciences, Orsay, France). Hybridization was performed in Quick Express Hyb solution (Clontech Laboratories, Palo Alto, CA) with an -32P-labeled human FIG1 RDA fragment or a
 -actin control probe (Clontech Laboratories) according to
the manufacturer's protocol.
DNA sequencing Plasmid DNA and PCR products were Taq cycle sequenced using the Applied Biosystems PRISM ready reaction Dye-dideoxy Terminator and Dye-Primer sequencing kits (Applied Biosystems, Courtaboeuf, France) and samples run on an ABI 377 DNA sequencer (Applied Biosystems).Quantitative RT-PCR analysis Total RNA (2 µg) was reverse transcribed with Superscript II (Life Technologies) in a final volume of 20 µL containing 300 ng random hexamers, according to the manufacturer's instructions. Following enzyme heat inactivation, cDNA was diluted 1:5 in water and stored at 20°C. FIG1 mRNA levels were measured by
real-time quantitative reverse transcription-polymerase chain reaction
(RT-PCR) using the Light Cycler (Roche Diagnostics, Meylan, France)
technology and normalized to the ribosomal S14 mRNA values
to control for RNA quality and reverse transcription efficiency. For
each real-time PCR run, cDNA was run in duplicate in parallel with 8 standard dilution samples and Karpas 1106 cDNA. FIG1 and
S14 standards were purified PCR products that were sequenced
and quantitated using a Genequant spectrophotometer (Amersham
Biosciences). Karpas 1106 cDNA was included in each PCR run to control
for PCR reproducibility among runs and allow relative expression to be
compared across all the tested samples. PCRs were performed in Light
Cycler capillaries, in a 20 µL volume containing 2 µL Light
Cycler-FastStart DNA Master SYBR Green I mix (Roche Diagnostics), 4 mM
final MgCl2 concentration, 0.5 µM FIG1 sense and antisense
primers (FIG1 sense: 5' TATGTGGTGGAGAAGGTG 3',
FIG1 antisense: 5' ATGCGGCTGTACTGGAGTC 3', purchased from Eurogentec, Serain, Belgium), or 0.2 µM S14 sense and
antisense primers (S14 sense: 5' GGCAGACCGAGATGAATCCTCA 3',
S14 antisense: 5' CAGGTCCAGGGGTCTTGGTCC 3', purchased from
Biotez GmbH, Berlin, Germany), and 2 µL cDNA diluted 1:3
(corresponding to 13 ng RNA). After preincubation and DNA denaturation
at 95°C for 8 minutes, 40 cycles of amplification (95°C for
10 seconds, 62°C for 5 seconds for FIG1, or 65°C for 5 seconds for S14, 72°C for 15 seconds) were performed and
PCR products were further denatured at 95°C, annealed at 70°C, and
slowly heated to 95°C to perform a melting curve analysis.
FIG1/S14 mRNA ratios were determined as mean FIG1 value / mean S14 value × 100.
Statistical analysis To test the differences between PMBL and NM-DLBL, we compared the FIG1/S14 mRNA ratios between these 2 categories of lymphomas using a Fisher exact test and a Mann-Whitney U test (Statview software; Abacus Concepts, Berkeley, CA).DNA extraction and Southern blot analysis DNA was extracted from MedB-1 and Ramos cells, from 5 PMBL and 5 NM-DLBL tumor samples, by proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation.26 After digestion with EcoRI, DNA fragments were electrophoresed in a 0.8% agarose gel in TAE buffer (40 mM Tris-acetate,1 mM EDTA, pH 8.3) and transferred onto a nylon N+ membrane (Amersham Biosciences). Hybridization was performed in Quick Express Hyb Solution (Clontech) with an-32P-labeled FIG1 probe, spanning
nucleotide 444 to 852 of FIG1 cDNA (according to AF 293462).
RDA identification of differential cDNA fragments RNA isolated from 5 PMBL and 5 NM-DLBL cases was pooled to generate the tester (PMBL) and driver (NM-DLBL) cDNA representations. After 2 rounds of subtractive hybridization and selective PCR amplification, the second difference product, DP2, showed several distinct bands that were resolved in an acrylamide gel (data not shown). Each of these bands was eluted from the gel and cloned into a pBluescript II KS plasmid. Inserts from these clones were used as probes to hybridize Southern blots of initial tester and driver representations, and sequenced when differentially expressed. We found that 14 DpnII fragments originating from 9 different genes were differentially expressed in PMBL versus NM-DLBL cDNA representations (Table 1).
Some of these genes, such as elongation factor 2 on chromosome 19pter-q12, and both complement C1r and CD163 on 12p13, were located in chromosomal regions showing frequent gains in PMBL.27,28 One of these fragments was homologous to the mouse interleukin
4-induced gene 1, identified as an IL-4 immediate-early response gene
in splenocytes.22 Specific and recurrent expression of the
human FIG1 mRNA in PMBL was studied by Northern blot
analysis. The FIG1 RDA fragment hybridized with 1.9-kb
transcripts, which were highly expressed in the 5 original PMBLs but
low or undetectable in the 5 original NM-DLBLs (Figure
1).
Structural features of human FIG1 cDNA and protein Human FIG1 cDNA sequence was determined by sequencing overlapping RT-PCR, 5'RACE, and 3'RACE fragments, and alignment of these sequences with the RDA fragment and human expressed sequence tags of the Unigene cluster Hs.380 444. The consensus sequence obtained comprised 1781 nucleotides (nt) and was identical to the sequence recently submitted to GenBank by Chu et al, except for a shorter 5' end (16 nt).29 This sequence
exhibited an open reading frame (ORF) of 1701 nt, with an ATG start
codon lying in a strong Kozak context (RnnatgG).30 The 3'
untranslated region of FIG1 was very short, the stop codon
being located within the variant polyadenylation signal ATTAAA (data
not shown).31 Alignment of the human cDNA sequences on the
human genome revealed that the human gene is located within a small
region (7 kilobase [kb]) of chromosome 19q13.33, and is composed of 8 exons, showing an exon/intron organization identical to the mouse gene.
Human FIG1 cDNA encodes a 567 amino acid (aa) protein that
contains 4 potential N-glycosylation sites, several potential protein kinase C, casein kinase II, tyrosine kinase, and cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP) protein kinase
phosphorylation sites, and 5 N-myristoylation sites. This protein
presents a putative signal peptide (aa 1-22) and a large flavin binding
amino-oxidase domain (aa 78-505; Figure
2). Human FIG1 protein is 80% homologous
to the mouse FIG1 protein in its 505 aa N-terminal part and completely
divergent in its 62 aa C-terminal part. Interestingly, human and mouse
FIG1 proteins show significant homology with the fish endoplasmic
reticulum lumenal L-amino acid oxidase (ERL-LAO, also named AIP for
apoptosis-inducing protein) and the snake venom Apoxin 1 (41%
and 33% identity, respectively; Figure 2), which both possess an
L-amino acid oxidase enzymatic activity and induce apoptosis through
H2O2 production.32-34
FIG1 mRNA expression pattern in normal human tissues and transformed cell lines In nonhematopoietic tissues, FIG1 transcripts were not detected in prostate, ovary, small intestine, and colon, but the FIG1 RDA probe hybridized with abundant 2.4-kb transcripts in the testis (Figure 3A). In normal lymphoid tissues, FIG1 transcripts were detected at very low levels in the thymus, spleen, tonsil, reactive lymph nodes, and resting tonsillar B cells (Figure 3B). As originally described in mice and recently in humans,22,29 RT-PCR experiments showed that FIG1 RNA is induced by IL-4 alone in tonsillar B cells (data not shown). Its expression was highly up-regulated when tonsillar B cells were activated with both IL-4 and CD40 ligand (Figure 3B).
We then studied FIG1 mRNA expression in a panel of human cell lines (Figure 3C). In B-cell lines, FIG1 transcripts were exclusively seen in the PMBL-derived B-cell lines Karpas 1106 and MedB-1, the latter showing a very high level of FIG1 mRNA expression. FIG1 mRNA expression was not detected in pro-B- (RS 4:11, Nalm16), pre-B- (697, Nalm6), Burkitt (Ramos), and the t(14;18) positive RL cell lines. Other hematopoietic (Jurkat, SUDHL1, Peer, DU528, HEL) and nonhematopoietic (HepG2, MZ2, Hela) cell lines were negative for FIG1 expression (data not shown). FIG1 mRNA expression in lymphoid malignancies FIG1 mRNA expression was first analyzed by Northern blot in a limited series of different low- and high-grade B-cell lymphoma entities. We selected for this analysis representative cases of lymphomas ranging from naïve B-cell-derived lymphomas to post-germinal center B-cell-derived lymphomas for which frozen material was available. This series included 2 chronic lymphocytic leukemias (CLLs), 2 mantle cell lymphomas (MCL), 2 Burkitt lymphomas (Bkt), 2 follicular lymphomas (FL), 2 marginal zone lymphomas (MZLs), 2 PMBLs, 4 NM-DLBLs including 2 NM-DLBLs used in the initial RDA experiments, and 2 plasmablastic lymphomas (PL-DLBLs). As shown in Figure 4A, FIG1 mRNA was highly expressed in PMBL and weakly expressed in the other B-cell lymphomas tested.
We then performed real-time quantitative RT-PCR analysis of
FIG1 gene expression in the normal and neoplastic samples
used in Northern blot experiments. We found that the
FIG1/S14 mRNA ratios evaluated with this technique were
parallel to the level of expression observed in Northern blot analysis.
Normal lymphoid tissues (spleen, thymus, lymph nodes) displayed
FIG1/S14 mRNA ratios inferior or equal to 10. The
FIG1/S14 mRNA ratio was equal to 1 in purified tonsillar B
cells and increased to 40 after IL-4+CD40L stimulation.
FIG1/S14 mRNA ratios varied from 12 to 101 in the 5 original
PMBLs and were less than 10 in the 5 original NM-DLBLs. All other
B-cell lymphomas tested displayed FIG1/S14 mRNA ratios less
than 10. We therefore decided to use a FIG1/S14 mRNA cutoff ratio of 10 to distinguish 2 categories of samples: those with high
FIG1 gene expression (FIG1/S14 mRNA ratio > 10) and those with low FIG1 mRNA levels (FIG1/S14
mRNA ratio We then extended our analysis to a larger series of 17 cases of PMBL and 18 cases of NM-DLBL, including the 5 PMBLs and 5 NM-DLBLs used in the RDA experiment. All but one PMBL (16/17) displayed high FIG1 gene expression, whereas most NM-DLBLs (12/18) exhibited low FIG1 mRNA levels (Figure 4B). Among the NM-DLBLs with high FIG1 mRNA levels, 5 were peculiar, because of extranodal origin (one spleen, one skin), suspected origin from the transformation of a follicular and marginal zone lymphoma, respectively (2 cases), or Epstein-Barr virus (EBV) association (one case). The difference between the PMBL and NM-DLBL groups proved significant (Fisher exact test based on the 2 category grading; P = .0003). Moreover, comparison of FIG1/S14 mRNA ratio as a continuous variable in PMBL versus NM-DLBL also demonstrated a significant difference (Mann-Whitney U test, P = .014). Southern blot analysis of FIG1 gene To study whether genomic rearrangements might account for FIG1 gene expression in PMBL, DNAs were extracted from PMBL and NM-DLBL tumor samples, subjected to EcoRI digestion, and analyzed by Southern blot. The blot was hybridized with a FIG1 PCR-generated probe spanning exons 5, 6, and 7. This probe hybridized with the expected 10-kb and 4.5-kb DNA fragments in all PMBL and NM-DLBL tumor samples, as well as in the MedB-1 and the Ramos B-cell lines (Figure 5). Hence, the increased expression of FIG1 mRNA in PMBL did not result from major genetic alterations. Furthermore, the signals correlated with the amount of DNA loaded in each lane, thereby ruling out overexpression related to gain of chromosomal material.
Using cDNA representational difference analysis, we
identified the FIG1 mRNA as being regularly expressed in
PMBL but rarely in NM-DLBL. FIG1 was initially identified in
the mouse as an immediate-early IL-4-inducible gene in B
splenocytes.22 This B-cell-specific gene was shown to be
induced in resting cells within 2 hours in response to IL-4 alone, but
not to IL-2, IL-5, or IL-6. This induction was recently demonstrated to
be STAT6 dependent.35 We have shown by Northern blot and
quantitative RT-PCR the high expression of FIG1 in
IL-4-activated human B-cells, in the PMBL-derived B-cell lines MedB-1
and Karpas 1106, and in PMBL as compared with other types of B-cell
lymphomas. Since genomic amplification of the FIG1 gene in
PMBL was ruled out by Southern blot analysis, these results point to a
possible role of the IL-4 signaling pathway in PMBL lymphomagenesis. It
was recently reported that IL-4 activation of the MedB-1 B-cell line
induced down-regulation of IgG/ Another explanation for high FIG1 expression in PMBL could be an activation of the IL-4 signaling pathway through oncogenic events. Oncogenic activation of cytokine signaling pathways in hematopoietic malignancies has already been reported. For example, IL-13 was shown to be secreted by Hodgkin lymphoma (HL) cell lines and to be expressed in Hodgkin and Reed-Sternberg cells in HL.37 IL-13 is thus believed to promote autocrine growth of the neoplastic population in classical Hodgkin lymphoma. Interestingly, JAK2 tyrosine kinase gene is amplified in MedB-1 and was found as part of an amplicon in one case of PMBL.15 As Jak-2 has been shown to be involved in the IL-4 and IL-13 signaling pathway in some nonhematopoietic cell lines,38 one may hypothesize that FIG1 up-regulation is related to abnormal JAK2 activity in PMBL. It is also possible that FIG1 expression in PMBL merely reflects the B-lymphocyte developmental stage at which malignant transformation occurred. FIG1 gene expression is induced in activated B cells. Thus, FIG1 gene expression in PMBL would favor the hypothesis that these lymphomas belong to the activated B-cell-like DLBL group,39 as already suggested by the presence of heavily mutated immunoglobulin genes without evidence of continuing mutational activity in both lymphoma groups.40,41 The protein encoded by the FIG1 gene shares significant homology with secreted apoptosis-inducing L-amino acid oxidases such as Apoxin 1 and ERL-LAO,32-34 suggesting that FIG1 protein might be an ectoenzyme. LAOs catalyze the oxidative deamination of various L-amino acids and produce ammonium and hydrogen peroxide (H2O2). In addition to its ability to induce apoptosis, H2O2 is becoming increasingly recognized as a signal-transducing molecule and appears to be involved in a broad spectrum of signaling pathways.42-44 Furthermore, recent studies have shown that some ectoenzymes are also involved in cellular adhesion.45,46 Thus, although the functions of the protein encoded by the FIG1 gene remain to be characterized, it is tempting to speculate that FIG1 activity might somehow be involved in PMBL lymphomagenesis. In conclusion, we demonstrate in this study the high expression of the IL-4-inducible gene FIG1 in PMBL. Our data suggest that the IL-4/IL-13 pathway may be of significant importance in PMBL lymphomagenesis. Exploration of FIG1 functional properties could give more insight into the oncogenic events involved in this distinct subtype of DLBLs.
We gratefully acknowledge the following pathologists who provided pathologic material: J. Brière, I. Abd Alsamad, A. M. Roucayrol. We thank N. Nio for expert assistance in statistical analysis. We also thank J. Marquet for providing resting and activated tonsillar B cells and S. Legouvello for help in real-time PCR experiments.
Submitted July 23, 2002; accepted November 6, 2002.
Prepublished online as Blood First Edition Paper, November 21, 2002; DOI 10.1182/blood-2002-07-2215.
Supported by a grant from the Association pour la Recherche contre le Cancer (no. 5530) and by the Association pour la Recherche Thérapeutique, Génétique et Immunologique dans les Lymphomes (cofinanced by Roche and Amgen).
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 U.S.C. section 1734.
Reprints: Karen Leroy, Département de Pathologie, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France; e-mail: karen.leroy{at}hmn.ap-hop-paris.fr.
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Top
Abstract
Introduction
immunophenotype.
), 17 cases of PMBL (
), and other B-cell
lymphomas (2 CLLs, 2 MCLs, 2 Bkt, 2 FLs, 2 MZLs; 
) are represented
in a scatter plot. The dashed line represents the cutoff ratio used to
distinguish high and low levels FIG1 gene expression.
10).
protein expression in a small subset
of tumor cells, suggesting that the lack of immunoglobulin expression
very frequently observed in PMBL could be due to extrinsic signals, one
of which might be IL-4.