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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1156-1164
Genes Encoding Tumor-Specific Antigens Are Expressed in Human Myeloma
Cells
By
Nicolas van Baren,
Francis Brasseur,
Danièle Godelaine,
Gérald Hames,
Augustin Ferrant,
Frédéric Lehmann,
Marc André,
Christophe Ravoet,
Chantal Doyen,
Giulio C. Spagnoli,
Marleen Bakkus,
Kris Thielemans, and
Thierry Boon
From Ludwig Institute for Cancer Research, Brussels, Belgium;
Unité de Génétique Cellulaire, Université
Catholique de Louvain, Brussels, Belgium; Cliniques Universitaires
Saint-Luc, Brussels, Belgium; Institut Jules Bordet, Brussels, Belgium;
Centre Hospitalier Notre-Dame et Reine Fabiola, Charleroi, Belgium;
Hôpital de Jolimont, Haine-St-Paul, Belgium; Cliniques
Universitaires de Mont-Godinne, Yvoir, Belgium; the Department of
Surgery, Research Division, University of Basel, Basel, Switzerland;
and Laboratorium voor Fysiologie, Vrije Universiteit Brussel, Brussels,
Belgium.
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ABSTRACT |
Genes of the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1
families encode antigenic peptides that are presented by HLA class I
molecules and that are recognized on human tumors by autologous
cytolytic T lymphocytes. These genes are expressed in many solid tumor
types but not in normal tissues, except male germline cells. Because the latter cells are devoid of HLA molecules, the derived antigens are
strictly tumor-specific and should constitute safe immunogens for
cancer immunotherapy. We detected a significant expression of these
genes in a high proportion of bone marrow samples from patients with
advanced multiple myeloma. This observation provides a basis for
clinical trials aimed at inducing a cellular immune response directed
at malignant plasma cells in advanced myeloma patients.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CONVENTIONAL chemotherapy or high-dose
chemotherapy followed by autologous hematopoietic stem cell
transplantation has improved the overall response rate and survival of
myeloma patients. However, most, if not all of the patients who achieve a complete remission after such treatment will ultimately relapse, showing that chemotherapy alone is unable to cure multiple
myeloma.1 Obviously, new approaches to treatment are needed.
There is increasing evidence that the immune system, and particularly T
lymphocytes, can target malignant plasma cells. Clinical responses in
myeloma patients treated with interleukin-2 (IL-2) have been
reported.2 After allogeneic bone marrow transplantation, T
lymphocytes from the donor can reject myeloma cells from the recipient,
eventually leading to long-term complete remissions. This
graft-versus-myeloma effect can also be induced by the reinfusion of
donor lymphocytes in myeloma patients who are in relapse after bone
marrow allografting.3,4 However, immune responses induced by IL-2 treatment or derived from the allogeneic graft are not myeloma-specific, and both are associated with significant toxicity. The efficiency and safety of immunotherapy against myeloma could therefore benefit from the identification of antigens present on
myeloma cells and absent on nonmalignant cells.
The monoclonal Ig produced by a B-cell-derived malignancy constitutes
a tumor antigen that is specific for this individual malignant clone.
Promising immunological and clinical responses were obtained by using
this immunogen in patients with B-cell lymphomas.5 In a
case of myeloma, the monoclonal Ig produced by the recipient's
malignant plasma cells was used to immunize the donor before allogeneic
bone marrow transplantation. This resulted in a detectable
anti-idiotype cellular immune response in both donor and allografted
recipient.6 In a recent report, 5 myeloma patients were
immunized with their respective purified monoclonal Ig in combination
with granulocyte-macrophage colony-stimulating factor (GM-CSF). An
idiotype-specific cellular immune response was reported in all
patients. One patient showed a decrease in the serum concentration of
the monoclonal component after immunization, suggesting a tumor
response to the vaccine.7
Another possible T-lymphocyte target present on myeloma cells is MUC1,
an immunogenic epithelial mucin present in an underglycosylated form on
breast, pancreatic, and ovarian carcinomas.8
Underglycosylated MUC1 was also detected on malignant plasma
cells,9 and anti-MUC1 cytolytic T lymphocytes (CTL) could
be obtained from the bone marrow of myeloma patients.10
Another category of antigens is encoded by genes of the MAGE
family. MAGE-A1 was initially isolated from a human melanoma cell line as a gene encoding an antigenic peptide presented to an
autologous CTL clone by HLA-A1 molecules.11 This gene is 1 of 12 closely homologous members of the MAGE-A family, all
located near the telomeric end of the long arm of chromosome
X.12 Genes MAGE-A1, A2, A3, A4, A6, A10, and
A12 are frequently expressed in many tumor types, such as
melanoma, bladder carcinoma, non-small cell lung carcinoma, head and
neck carcinoma, and esophagus squamous cell carcinoma, but are silent
in normal tissues except testis and, in some cases, placental
trophoblast cells. Immunohistochemistry studies have shown that, in
testis, MAGE proteins are present in spermatogonia.13 These
cells are known to lack expression of HLA molecules and are therefore
unable to present peptides to CTL. Because this is also true for the
trophoblast, the MAGE-derived antigens are strictly
tumor-specific.
BAGE and GAGE-1 were also identified as genes encoding
antigens recognized by autologous CTL on a melanoma cell
line.14,15 They share the same pattern of expression as
MAGE and are therefore referred to as MAGE-type genes.
Another MAGE-type gene is LAGE-1. It was isolated by
representational difference analysis as a gene expressed in a melanoma
cell line but not in normal skin.16 It is homologous to
NY-ESO-1, a gene that encodes an antigen recognized by
autologous antibodies isolated from a patient with esophageal squamous
cell carcinoma.17 Both are expressed in many solid tumors
and in testis.
The MAGE-A1 gene becomes activated in tumor cells as a result
of demethylation of CpG dinucleotides in its promoter. In
nontumoral cells, the methylation of these sites inhibits the binding
of activating transcription factors and results in gene
silencing.18 Expression of the MAGE-type genes can
be induced experimentally in nontumoral growing cells by incubation
with the demethylating agent 5-aza-2'-deoxycytidine, which shows that
DNA demethylation is the common mechanism that accounts for aberrant
expression of these genes.
Antigens encoded by MAGE-type genes may be particularly
suitable as targets for immunotherapy, because they are strictly
tumor-specific and are shared by many different tumor types. In the
present study, we investigated the expression of genes of the
MAGE-A, GAGE, and LAGE-1/NY-ESO-1 families and of gene
BAGE in bone marrow or blood samples from patients with
multiple myeloma or monoclonal gammopathy of undetermined significance
(MGUS). We also tested the expression of gene PRAME, which
encodes an antigen recognized by a CTL clone on a melanoma cell line
and is very frequently expressed in tumors of several types, as well as
in testis. It is not as tumor-specific as the MAGE-type genes,
because there is a low level of expression in the endometrium, ovary,
and adrenals.19 Its mRNA has also been detected in acute
leukemias and in a few samples of lymphoma and myeloma.20
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MATERIALS AND METHODS |
Tumor sample collection and processing.
Cells were collected from normal donors or from patients with MGUS or
multiple myeloma by bone marrow aspiration. Samples from myeloma
patients were collected before they received any treatment or at
distance from previous chemotherapy. Cytologic examination confirmed
that all myeloma samples contained malignant plasma cells. The
mononuclear cells were purified by Lymphoprep (Nycomed, Oslo, Norway)
density gradient centrifugation and were washed twice with Iscove's
medium (GIBCO Laboratories, Green Island, NY) containing 10% fetal
calf serum (FCS; GIBCO) and twice with phosphate-buffered saline (PBS).
After a final centrifugation, the dry cell pellet was stored at
 80°C until needed for RNA extraction.
Reverse transcription-polymerase chain reaction (RT-PCR) assay.
RNA extraction and RT-PCR amplifications were performed as described
previously,21 with slight modifications. Briefly, cDNA was
synthesized from 2 µg of total RNA by extension with oligo(dT) primer
and 200 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (GIBCO) at 42°C for 90 minutes. PCR amplification was
performed on 1/40th of the cDNA solution with 0.625 U of Taq DNA
polymerase (Takara, Shiga, Japan) in a final volume of 25 µL. The PCR conditions and primers were as
follows: MAGE-A1: 94°C for 4 minutes and 30 cycles of 94°C
for 1 minute, and 72°C for 2 minutes, primers
5'-CGGCCGAAGGAACCTGACCCAG-3' and 5'-GCTGGAACCCTCACTGGGTTGCC-3'; MAGE-A2: 94°C for 4 minutes and 30 cycles of 94°C for 1 minute, 67°C for 1 minute, and 72°C for 1 minute, primers
5'-AAGTAGGACCCGAGGCACTG-3' and 5'-GAAGAGGAAGAAGCGGTCTG-3';
MAGE-A3: 94°C for 4 minutes and 30 cycles of 94°C for 1 minute and 72°C for 2 minutes, primers 5'-TGGAGGACCAGAGGCCCCC-3' and
5'-GGACGATTATCAGGAGGCCTGC-3'; MAGE-A4: 94°C for 4 minutes and
30 cycles of 94°C for 1minute, 68°C for 1 minute and 72°C for 1 minute, primers 5'-GAGCAGACAGGCCAACCG-3' and 5'-AAGGACTCTGCGTCAGGC-3';
MAGE-A6: 94°C for 4 minutes and 30 cycles of 94°C for 1 minute, 70°C for 2 minutes, and 72°C for 2 minutes, primers
5'-TGGAGGACCAGAGGCCCCC-3' and 5'-CAGGATGATTATCAGGAAGCCTGT-3'; MAGE-A10: 94°C for 4 minutes and 30 cycles of 94°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute, primers
5'-CACAGAGCAGCACTGAAGGAG-3' and 5'-CTGGGTAAAGACTCACTGTCTGG-3';
MAGE-A12: 94°C for 4 minutes and 32 cycles of 94°C for 1 minute, 62°C for 2 minutes, and 72°C for 3 minutes, primers
5'-CGTTGGAGGTCAGAGAACAG-3' and 5'-GCCCTCCACTGATCTTTAGCAA-3'; BAGE: 94°C for 4 minutes and 30 cycles of 94°C for 1 minute, 62°C for 2 minutes, and 72°C for 2 minutes, primers
5'-TGGCTCGTCTCACTCTGG-3' and 5'-CCTCCTATTGCTCCTGTTG-3';
GAGE-1/2: 94°C for 4 minutes and 30 cycles of 94°C for 1 minute, 56°C for 2 minutes, and 72°C for 2 minutes, primers
5'-GACCAAGACGCTACGTAG-3' and 5'-CCATCAGGACCATCTTCA-3'; GAGE-3/6: 94°C for 4 minutes and 30 cycles of 94°C for 1 minute, 58°C for 2 minutes, and 72°C for 2 minutes, primers
5'-GACCAAGGCGCTATGTAC-3' and 5'-CCATCAGGACCATCTTCA-3'; LAGE-1:
94°C for 4 minutes and 30 cycles of 94°C for 1 minute, 62°C for 1 minute, and 72°C for 1 minute, primers 5'-GCAGGATGGAAGGTGCCC-3' and
5'-CTGGCCACTCGTGCTGGGA-3'; NY-ESO-1: 94°C for 4 minutes and
30 cycles of 94°C for 1 minute, 62°C for 1 minute, and 72°C for 1 minute, primers 5'-CCCCACCGCTTCCCGTG-3' and 5'-CTGGCCACTCGTGCTGGGA-3';
PRAME: 94°C for 4 minutes and 30 cycles of 94°C for 1 minute, 64°C for 1 minute, and 72°C for 1 minute, primers
5'-CTGTACTCATTTCCAGAGCCAGA-3' and
5'-TATTGAGAGGGTTTCCAAGGGGTT-3';  -ACTIN: 94°C for 4 minutes and 21 cycles of 94°C for 1 minute, 68°C for 1 minute, and
72°C for 1 minute, primers 5'-GGCATCGTGATGGACTCCG-3' and
5'-GCTGGAAGGTGGACAGCGA-3'.
Cycling was concluded with a final extension step of 15 minutes at
72°C. Each primer was chosen in a different exon to avoid false-positives caused by DNA contamination of the RNA preparation. Assessment of the PCR product was performed visually on an ethidium bromide-stained agarose gel by comparing the intensity of the band with
that resulting from RT-PCR performed on serial dilutions (1:1, 1:3,
1:9, and 1:27) of the RNA from 1 of 3 tumor cell lines (2 melanomas and
1 sarcoma) used as a positive control and reference for the level of
expression. These cell lines were MZ2-MEL (all MAGE, BAGE, and
GAGE PCR except MAGE-A4 and MAGE-A12), LB23-SARC (MAGE-A4
PCR), and LB373-MEL (MAGE-A12, LAGE-1, NY-ESO-1, and PRAME PCR).
Samples were scored +++, ++, +, or ± if the amount of the amplified
product was equal to or greater than that obtained with the 1:1, 1:3,
1:9, and 1:27 dilutions of the reference RNA, respectively. Lower
levels of expression were scored negative. An expression level of the
-ACTIN gene comparable with that of the positive control was
obtained with each sample.
Lysis assay.
All cell lines were grown in RPMI-1640 medium (GIBCO) supplemented with
5% FCS (EJM and U266) or 10% FCS (MZ2-MEL and SK23-MEL). The
anti-MAGE-3.A1 CTL clone 434/1 was derived from the blood of an HLA-A1
patient with hemochromatosis after repetitive stimulation with
autologous phytohemagglutinin (PHA)-stimulated T cells incubated with
the MAGE-3.A1 peptide. The anti-MAGE-3.A2 CTL clone 297/22 was
obtained similarly from an HLA-A2 patient.22 Chromium
release assay was performed as described previously.23
Briefly, target cells were labeled with 51Cr, washed, and
dispensed into microwell plates at 1,000 cells per well in Iscove's
medium supplemented with 10% human serum and with L-arginine (116 mg/L), L-asparagine (36 mg/L), and L-glutamine (216 mg/L), which is
further referred to as complete medium. CTL was added at increasing
effector-to-target ratios. The cells were centrifuged and incubated at
37°C for 4 hours, and chromium release was determined by measuring
the radioactivity in the supernatant. The myeloma cell lines were also
tested for their ability to stimulate the production of tumor necrosis
factor (TNF) by the CTL clones, as described previously.24
Briefly, 5,000 CTL were added to microwells containing 10,000 target
cells in 150 µL of complete medium supplemented with 25 U/mL of IL-2.
After overnight incubation, the supernatant was collected and its TNF
content was measured by testing its cytotoxic effect on WEHI-164 clone
13 cells in an MTT colorimetric assay.
Immunocytochemistry.
Myeloma cell lines EJM, U266, and Fravel and mononuclear cells isolated
from the bone marrow of patient no. 43 were washed in Tris-buffered
saline (TBS) and cytospun at 500 rpm for 4 minutes on microscope slides
(105 cells per slide). Mononuclear cells isolated from the
bone marrow of patient no. 21 were smeared on microscope slides. All of
the slides were air-dried at room temperature, wrapped in aluminium foil, and stored at 80°C until needed. They were fixed in 10% buffered formalin at room temperature for 10 minutes and then washed in
TBS for 2 minutes. The fixed slides were incubated with the anti-MAGE
hybridoma supernatant 57B,25 either undiluted (patient no.
21) or diluted 1/10 (others) at 4°C for 18 hours, or with an
isotype-matched irrelevant monoclonal antibody. They were further
incubated with biotinylated antimouse Igs and alkaline phosphatase-conjugated streptavidin (LSAB+; Dako, Glostrup, Denmark), using New Fuchsin as a chromogen. Sections of
formalin-fixed, paraffin-embedded gut biopsies from patient no. 29 were
heated, after deparaffinization, in a 1,500 W microwave oven twice for 5 minutes in citrate buffer for antigen retrieval (Dako). They were
washed in TBS containing 0.05% Tween 20 for 1 minute, incubated with
antibody 57B diluted 1/10 or with the irrelevant antibody, at room
temperature for 1 hour, and then incubated with a peroxydase-conjugated polymer backbone carrying antimouse Igs (En Vision; Dako) with AEC as
chromogenic substrate.
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RESULTS |
Expression of MAGE-type genes in bone marrow samples from MGUS or
myeloma patients.
We used RT-PCR to analyze 44 bone marrow samples from patients with
MGUS (n = 6) or myeloma (n = 38) for the expression of 7 genes of
the MAGE-A family, the gene BAGE, 2 subgroups of very closely related genes of the GAGE family, the gene
LAGE-1, the gene NY-ESO-1, and the gene PRAME.
Two samples were obtained from the peripheral blood of patients with
plasma cell leukemia (Table 1 and Fig
1). When considering only +++, ++, and + scores, all of the samples from patients with MGUS and with stage I and
stage II myeloma were found negative, except for 1 MGUS sample that showed significant expression of gene LAGE-1. In contrast, a
majority of the samples from the stage III myeloma patients expressed
at least 1 of these genes. When considering stage III myelomas and plasma cell leukemias, the most frequently expressed genes were LAGE-1 (52%), PRAME (48%), and GAGE (41%),
followed by NY-ESO-1 and MAGE-A6 (31%),
MAGE-A1 and MAGE-A3 (28%), MAGE-A2 and
MAGE-A4 (17%), MAGE-A12 and BAGE (14%), and
finally MAGE-A10 (7%). In total, 38% of these samples were
positive for at least 1 of the MAGE-A genes, and 62% were
positive for at least 1 of the MAGE-type genes. In addition, we
tested 7 bone marrows from normal donors. All were negative (data not
shown).
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Table 1.
Detection of mRNA From the Indicated Genes by RT-PCR in
Bone Marrow or Blood Samples From Patients with Monoclonal Gammopathy
of Undetermined Significance (MGUS), Multiple Myeloma, and Plasma Cell
Leukemia (PCL), Classified According to Durie and
Salmon31
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| Fig 1.
RT-PCR amplification products of the indicated genes
obtained with bone marrow samples from 8 different patients with MGUS
(no. 5), myeloma stage I (no. 12), or myeloma stage III (nos. 21 through 39). C+, positive control melanoma line (MZ2-MEL for MAGE-A3,
GAGE-3/6, and -ACTIN; LB373-MEL for LAGE-1 and PRAME). C,
negative control (no RNA present in the RT reaction). MW, molecular
weight marker is SmartLadder (Eurogentec, Seraing,
Belgium). Amplifications of MAGE-A3, GAGE-3/6, PRAME,
and -ACTIN transcripts give unique bands of 725, 244, 561, and 615 bp, respectively. Amplification of LAGE-1 transcript gives 2 bands that
correspond to fully spliced (399 bp) and partially spliced (628 bp)
mRNA, respectively, and a band of approximately 600 bp representing
heteroduplexes formed during PCR between the products amplified from
the partially and the fully spliced cDNAs.
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Recognition of myeloma cells by anti-MAGE CTL.
We used 2 anti-MAGE-A3 CTL clones, 1 restricted by HLA-A1 and the
other by HLA-A2. Only 2 myeloma cell lines with proper HLA type and
MAGE expression were available. The anti-MAGE-A3.A1 CTL lysed myeloma
EJM as well as control melanoma target cells. The anti-MAGE-A3.A2 CTL
showed weak but significant lysis against myeloma U266, but this CTL
appears to recognize a poorly processed peptide and lyses weakly most
melanoma cell lines (Fig 2A). A more
sensitive TNF release assay demonstrated recognition of the 2 myeloma
cell lines by the appropriate anti-MAGE CTL (Fig 2B).
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| Fig 2.
(A) Lytic activity of anti-MAGE-A3.A1 CTL clone 434/1
and anti-MAGE-A3.A2 CTL clone 297/22 against myeloma cell lines EJM
and U266. Expression of gene MAGE-A3 was assessed by RT-PCR by using
the same procedure as for the primary myeloma samples. (B) TNF-
release by the same CTL clones after incubation with the same myeloma
cell lines.
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Staining of myeloma cells with a MAGE-specific monoclonal antibody.
The monoclonal antibody 57B was initially reported to recognize the
MAGE-A3 protein.25 We have observed that this antibody recognizes not only the MAGE-A3 protein, but also the MAGE-A1, A2, A4,
A6, and A12 proteins. It stained the MAGE-expressing human myeloma cell lines EJM and U266 (Fig 3A and
B) and did not stain the
MAGE-negative myeloma cell line Fravel (Fig 3C). We tested samples of a few patients with a MAGE-expressing myeloma,
recorded in Table 1. Patient no. 29 developed skin and gut
plasmacytomas during the course of a stage III myeloma treated with
chemotherapy. His bone marrow was found to be positive for expression
of all the genes tested in this report. A gut biopsy section was
stained with antibody 57B. The gut wall showed a massive infiltration by malignant plasma cells (Fig 3D). A similar result was obtained with
a cutaneous plasmacytoma from the same patient (data not shown). In
both samples, all the malignant plasma cells were homogeneously stained, whereas the normal cells were negative. A bone marrow smear
from patient no. 21, who had an untreated MAGE+ stage III
myeloma, was stained with the same antibody. Here also, all of the
malignant plasma cells were positive (Fig 3E). A cytospin performed
with bone marrow mononuclear cells from patient no. 43, who had a
MAGE+ myeloma in relapse after 2 autologous bone
marrow transplantations, was stained with 57B. Approximately 30% of
all the malignant plasma cells were positive, whereas normal bone
marrow cells and the remaining myeloma cells were negative (Fig 3F).
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| Fig 3.
Immunocytochemistry performed on myeloma samples with
anti-MAGE monoclonal antibody 57B. (A) MAGE+ myeloma cell line EJM.
(B) MAGE+ myeloma cell line U266. (C) MAGE myeloma cell line
Fravel. (D) Gut biopsy from patient no. 29, a patient with MAGE+
stage III myeloma treated by chemotherapy, who developed skin and gut
plasmacytomas. The cells stained in brownish red are malignant plasma
cells infiltrating the gut mucosa. The epithelium is indicated by an
arrow. (E) Bone marrow smear from patient no. 21, a patient with
MAGE+ stage III myeloma. All the plasma cells are stained in red. (F)
Bone marrow cytospin from patient no. 43, who had a myeloma in relapse
after 2 autografts. Some malignant plasma cells are stained in red. The
arrows indicate 2 unstained myeloma cells. The slides incubated with
the isotype-matched irrelevant monoclonal antibody remained negative,
as did cytospins of MAGE-negative bone marrows from stage III myeloma
patients incubated with antibody 57B (data not shown).
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DISCUSSION |
Our results show that MAGE-type genes are expressed in the bone
marrow of myeloma patients. There is an obvious correlation between
expression of these genes and the stage of the disease, because they
are almost always silent in MGUS and stage I and II myelomas and are
frequently activated in stage III myelomas. A similar correlation has
already been observed in melanoma, in which metastases were found to be
more frequently positive for MAGE expression than primary
tumors26 and in bladder cancer, in which MAGE was
expressed more frequently in infiltrative tumors than in local
tumors.21 In addition, as in many solid tumors, several of
these genes are frequently coexpressed in individual myeloma samples.
Our observations indicate that myeloma cells may frequently present
tumor-specific shared antigens. It has been shown previously that
myelomas have a high level of expression of HLA class I molecules and
that they have the capacity to present antigenic peptides to T
lymphocytes and to activate these cells. In addition, they often carry
functional HLA class II molecules.27 Our
immunocytochemistry data confirm the presence of MAGE proteins in the
cytoplasm of malignant plasma cells from MAGE-expressing
myeloma bone marrows. Our lysis assays show that a
MAGE-expressing myeloma cell line can be killed by a CTL clone
recognizing a MAGE epitope. Immunotherapy with tumor-specific shared
antigens therefore constitutes a possible new treatment modality
against stage III myeloma.
Several antigenic peptides that are derived from the MAGE, BAGE, GAGE,
NY-ESO-1, and PRAME proteins and that are presented to CTL by HLA class
I molecules have been described. Table 2 gives a prediction of their frequency in advanced myeloma. It is quite
probable that many additional antigens derived from the same proteins
and from LAGE-1 still have to be identified. Thus, even if the
probability of having a given antigen on an individual myeloma is low,
many different antigens should be present simultaneously. For
immunotherapy, the use of multiple immunogens is important, because it
increases the probability of inducing a specific immune response and
reduces the risk of tumor escape by selection of antigen-loss variants.
Multiple immunogens can be delivered as a combination of peptides or as
a recombinant protein. They can also be provided as a defective virus
containing a gene or several minigenes coding for selected epitopes.
Clinical trials aimed at evaluating MAGE-derived immunogens against
solid tumors, mainly melanomas, are ongoing. In a recent report,
immunization of advanced melanoma patients with the MAGE-3.A1 peptide
led to objective regressions of metastases in 7 of 25 patients with
measurable disease who completed the treatment. No toxicity was
observed. However, no specific CTL response could be detected in the
blood of a subset of patients, including 2 who showed tumor
regressions.28 The encouraging clinical observation deserves further investigation. It is hoped that increased frequency of
tumor regressions and correlation with measurable immune response will
be achieved by using better immunogens, such as recombinant proteins or
viruses, by associating these immunogens with immunological adjuvants
or immunostimulatory cytokines, and by developing improved assays for
measuring antigen-specific CTL responses.
Myeloma might represent a valuable model to study the immunological and
clinical responses to vaccination with MAGE-type immunogens. It is a
chronic, incurable disease whose evolution can be monitored easily.
Moreover, myeloma bone marrows may constitute a reproducible source of
tumor-infiltrating lymphocytes, allowing for a more precise assessment
of CTL responses.
Our observations also have an implication with respect to the follow-up
of advanced myeloma patients treated by chemotherapy. Given that genes
MAGE, BAGE, GAGE, LAGE-1, and NY-ESO-1 are transcribed in myeloma cells but not in normal nongerminal tissues, including hematopoietic cells, the presence of the corresponding mRNA can be
considered as a tumor-specific marker. The same applies to PRAME, because it is not expressed in normal bone marrow or
peripheral blood leukocytes. Thus, the high sensitivity and specificity
of RT-PCR could allow the detection of a very low proportion of myeloma cells in the bone marrow or in the blood. This would be particularly useful to assess the response to high-dose chemotherapy, to detect residual disease in patients with cytological remission, and to detect
early relapse in these patients. Moreover, the detection of low
expression levels of MAGE-type mRNA in bone marrow from patients with
MGUS and stage I and stage II myeloma may predict the evolution towards
more advanced disease. It can be deduced from Table 1 that the
detection of all the samples that express at least 1 of the
tumor-specific genes analyzed in this report can be performed by RT-PCR
using only primers specific for GAGE, LAGE-1, and
PRAME.
The expression of the MAGE-type genes in tumors has been
demonstrated to be linked to overall DNA demethylation. Because almost 80% of stage III myelomas tested in our series expressed at least 1 of
these genes or PRAME, we consider it likely that overall DNA
demethylation occurs in many advanced myelomas. Genome-wide demethylation acquired by a few tumor cells may result in transcription of oncogenes that would otherwise remain silent. Their aberrant activation could confer a growth advantage to these cells as compared with normally methylated cells. It is unclear which oncogenes could
become activated in myeloma as a result of demethylation. Among
possible candidates, c-MYC is known to be frequently
overexpressed in malignant plasma cells. In some myelomas, its
overexpression seems to be a consequence of a fusion with the IgH locus
in the t(8;14) translocation.29 In 1 report, a CpG site in
the third exon of the c-MYC gene was found to be demethylated
in 5 myeloma cell lines that had a significant overexpression of
c-MYC as compared with normally methylated cells.30
However, a link between demethylation and c-MYC overexpression
remains to be demonstrated in fresh myeloma cells. It should be
mentioned that the function of the MAGE, BAGE, GAGE, LAGE-1, NY-ESO-1,
and PRAME proteins is unknown, and their involvement in tumor
progression remains unproven. The activation of these genes may be a
neutral side effect of a global demethylation process that activates
other genes involved in tumor progression.
It is important to note that, in 1 of the 3 samples analyzed by
immunocytochemistry, only a subset of the myeloma cells were stained by
the anti-MAGE antibody. This observation is consistent with the
acquisition of DNA demethylation by a few cells that will grow faster
and then progressively overwhelm the others. Targeting these more
tumorigenic cells by specific CTL might remain a valuable aim, because
the remaining myeloma cells would probably show less
aggressiveness. Immunization of stage I and II myeloma patients to prevent the emergence of demethylated myeloma cells and the
resulting progression to more aggressive disease should also be considered.
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ACKNOWLEDGMENT |
The authors are grateful to M. Hérin and J.-M. Scheiff for
reviewing the immunohistochemistry data. We thank C. Mondovits, B. Tollet, and M. Swinarska for excellent technical assistance; S. Mapp
for his help in the preparation of the manuscript; and P. Coulie for
helpful discussions.
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FOOTNOTES |
Submitted January 18, 1999; accepted April 21, 1999.
Supported by the Belgian Programme on Interuniversity Poles of
Attraction initiated by the Belgian State, Prime Minister's Office,
Science Policy Programming, and by grants from the Association contre
le Cancer (Brussels, Belgium), from the BIOMED2 programme of the
European Community, from the Fonds J. Maisin (Belgium), from
CGER-Assurances and VIVA (Brussels, Belgium), from the Fonds National
de la Recherche Scientifique (TELEVIE grants) (Brussels, Belgium), and
from the Swiss National Fonds Grant No. 31-45560.95.
The publication costs of this
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ABSTRACT
INTRODUCTION