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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3530-3533
NEOPLASIA
From INSERM U119 and Unité d'Immunologie des Tumeurs,
Institut Paoli Calmettes, Marseilles, France; the Ludwig Institute for
Cancer Research, Brussels, Belgium; Unité de
Génétique Cellulaire, Université catholique de
Louvain, Brussels, Belgium; Service d'Anatomopathologie, Institut
Paoli Calmettes, Marseilles, France; Service d'Anatomopathologie,
Cliniques Universitaires Saint-Luc, Brussels, Belgium; ETS
Isère-Savoie and Research Group on Lymphoma, Unité UPRES
2021, Grenoble, France; the Department of Surgery, Research Division,
University of Basel, Switzerland; and Service de Pédiatrie et
d'Hématologie Pédiatrique, CHU Timone, Marseilles,
France.
Genes of the MAGE-A family are expressed in several
types of solid tumors but are silent in normal tissues with the
exception of male germline cells, which do not carry HLA
molecules.Therefore, peptides encoded by MAGE-A genes are
strictly tumor-specific antigens that can be recognized by CTL and
constitute promising targets for immunotherapy. The expression of 6 genes of the MAGE-A family was tested with reverse
transcriptase-polymerase chain reaction in lymphoma samples.
Among 38 samples of non-Hodgkin lymphoma, 1 anaplastic large
cell lymphoma expressed genes MAGE-A1, -A2, -A3, -A4, and
-A12, and 1 lymphoepithelioid T-cell lymphoma expressed gene
MAGE-A4. Five of 18 samples (28%) from patients with Hodgkin disease expressed gene MAGE-A4. In tissue sections, staining by a monoclonal antibody that recognizes the MAGE-A4 protein was observed in 11 of 53 samples (21%) from patients with Hodgkin disease.
In the positive samples, the Reed-Sternberg cells were strongly stained
whereas the surrounding cells were not. These results indicate that
Hodgkin disease may be a target for specific immunotherapy involving
MAGE-A4 antigens.
(Blood. 2000;95:3530-3533)
Intensive chemotherapy and bone marrow
transplantation have improved the disease-free survival of patients
with non-Hodgkin lymphoma (NHL) and Hodgkin disease (HD). However, the
relapse rate remains important, particularly in NHL. Clinical
experience with allogeneic bone marrow transplantation demonstrated
that immune-mediated graft-versus-lymphoma reactions may be induced in
NHL patients, a situation that may be analogous to the
graft-versus-leukemia (GVL) effect that participates in the cure of
leukemia patients.1-6 Immunotherapy involving the
administration of immunostimulatory cytokines or donor lymphocytes
seemed to improve the clinical outcome of a few lymphoma
patients.7,8 However, optimizing the antilymphoma effect of
allogeneic reactions is clearly difficult, considering the mortality
and morbidity associated with graft-versus-host disease. New
immunotherapeutic tools would be necessary, at least as adjuvant
treatment, to control residual disease.
Tumor-specific antigens consisting of peptides presented to autologous
cytotolytic T lymphocytes by class I human leukocyte antigens (HLAs)
may constitute targets for specific immunotherapy.9 Several
types of antigens present on leukemia, lymphoma, or melanoma cells have
been described: chimeric proteins resulting from gene translocations
(for example, PML-RAR An interesting category of tumor antigens is encoded by genes such as
MAGE, BAGE, GAGE, or SSX, which are
expressed in many types of solid tumors but are silent in normal
tissues with the exception of male germinal cells.9,17-19
Because these cells do not carry HLA molecules, they cannot present
antigenic peptides. Antigens encoded by such "cancer/testis"
genes are therefore strictly tumor-specific and can be used safely in
immunotherapy trials. Regressions of metastatic melanoma lesions were
reported after immunization with an antigenic peptide encoded by gene
MAGE-A3 and presented by HLA-A1 molecules.20
MAGE-A is 1 of the 4 known MAGE gene families, and it
comprises 12 genes. We reported that gene MAGE-A1 was silent in
bone marrow or blood samples from 48 leukemia patients.21
The only hematologic malignancies in which MAGE gene expression
has been detected are adult T-cell acute lymphoblastic
leukemia and multiple myeloma.22-24 Among
the members of the SSX genes family, SSX-2 was shown to
be expressed in 4 of 11 NHL samples. Here we report on the analysis of
MAGE-A gene expression in lymph node biopsies from lymphoma patients.
Control cell lines
Normal lymphoid cells
Patients Fifty-six lymphoma patients were included in the study: 38 with NHL and 18 with HD. Diagnosis was based on conventional morphologic examination of paraffin-embedded material, fixed with formalin or Bouin's fluid. The slides were examined by 2 pathologists (L.X. and I.T.) and, when required, diagnoses were refined with immunohistochemistry using monoclonal antibodies (MAbs) recognizing B cells, T cells or Reed-Sternberg cells. According to the REAL classification,25 these lymphoid neoplasms were distributed as follows: 29 B-cell NHLs (2 small lymphocytic lymphomas, 5 mantle cell lymphomas, 14 follicle center lymphomas, 1 marginal zone B-cell lymphoma, 6 diffuse large B-cell lymphomas, and 1 plasmacytoma/plasma cell lymphoma), 9 T-cell NHLs (3 precursor T-lymphoblastic lymphomas, 5 anaplastic large cell lymphomas, and 1 lymphoepithelioid peripheral T-cell lymphoma), and 18 HD samples: 2 (HD1 and HD2) of type I (lymphocytic predominance), 8 (HD3-10) of type II (nodular sclerosis), 7 (HD11-17) of type III (mixed cellularity), and 1 (HD18) of type IV (lymphocyte depletion). Fifty-three HD samples, including 3 of type I, 34 of type II, 14 of type III, and 2 of type IV, were used for the immunohistochemical analysis. HD samples from 2 HD patients (HD3 and HD4) belonged to both series (RT-PCR analysis and immunohistochemistry).Lymph node biopsies Lymph nodes were either frozen in liquid nitrogen immediately after surgical excision, or they were minced to obtain a suspension of cells that were frozen in RPMI (GIBCO BRL, Gaithersburg, MD) with 37.5% (vol/vol) fetal calf serum (GIBCO BRL) and 10% dimethyl sulfoxide and stored in liquid nitrogen. For patient HD3, cryopreserved lymph node cells were thawed, treated by trypsin ethylenediaminetetraacetic acid, and separated by positive immunoselection with CD30 MAb and goat antimouse magnetic beads to obtain fractions enriched or depleted in CD30+ cells before RNA extraction.RT-PCR assay Total cellular RNA was isolated by the guanidine-isothiocyanate/cesium chloride procedure26 or with TRIzol (GIBCO BRL). Complementary DNA (cDNA) from 2 µg of total RNA was obtained by incubation at 42°C for 90 minutes with oligo(dT) primer and 200 units of Moloney murine leukemia virus RT (GIBCO BRL). PCR amplification was performed on 2.5% of the cDNA with 0.625 units of Taq DNA polymerase in a final volume of 25 µL. Reaction mixtures were heated at 94°C for 4 minutes and subjected to amplification for 30 (MAGE-A1 to -A6) or 32 cycles (MAGE-A12) consisting of 1 minute at 94°C; 1 minute (2 minutes for MAGE-A6 and -A12) at 72°C (MAGE-A1 and -A3), 70°C (MAGE-A6), 68°C (MAGE-A4), 67°C (MAGE-A2), or 62°C (MAGE-A12); and 1 minute (2 minutes for MAGE-A6 or 3 minutes for MAGE-A12) at 72°C. Final extension was obtained with 15 minutes at 72°C. The MAGE-specific primers were described previously,18,27 except for MAGE-A12: 5'-GCCCTCCACTGATCTTTAGCAA-3' (exon 3)18 and 5'-CGTTGGAGGTCAGAGAACAG-3' (exon 1). Each primer was chosen in a different exon to avoid false positives caused by DNA contamination of the RNA preparation. Samples with degraded RNA were excluded by testing the expression of the -actin gene. The amounts
of amplified products were evaluated visually on ethidium
bromide-stained agarose gels and identified by comparing the size of
the band with the molecular weight marker  X174 (GIBCO BRL). Bands of
test samples were compared with those resulting from RT-PCR performed on serial dilutions (1:1, 1:3, 1:9, 1:27) of RNA from a positive control cell line: MZ2-MEL-3.0 for MAGE-A1, -A2, -A3, -A6;
LB23-SARC for MAGE-A4; and LB373-MEL for MAGE-A12.
Immunohistochemistry Lymph node biopsy specimens from HD were formalin-fixed and embedded in paraffin. Immunohistochemical detection of the MAGE-A4 protein was performed on paraffin sections previously heated for 10 minutes in the ChemMate Buffer for Antigen Retrieval (DAKO, Trappes, France) with the mouse MAb 57B.28 The positive control was a sample of normal testis, whereas negative control consisted of replacing antibody 57B by an irrelevant isotype-matched MAb. The slides were processed using the alkaline phosphatase LSAB+ detection kit (DAKO) and the New Fuschin Substrate System (DAKO), according to the manufacturer's recommendations, and counterstained with hematoxylin.
RT-PCR analysis of MAGE-A expression in lymph nodes from NHL patients We tested 38 samples corresponding to various types of NHL. None of the 29 B-cell lymphomas expressed any of the 6 MAGE-A genes that were tested, whereas 2 of the 9 T-cell lymphomas were positive for at least 1 MAGE-A gene (Table 1). One anaplastic large-cell lymphoma expressed genes MAGE-A1, -A2, -A3, -A4, and -A12 at a significant level, whereas expression of gene MAGE-A6 was not detected. A low amount of MAGE-A4 transcript was detected in 1 sample of lymphoepithelioid peripheral T-cell lymphoma (Figure 1).
RT-PCR analysis of MAGE-A expression in lymph nodes from HD patients Eighteen samples were tested. Gene MAGE-A4 was found to be expressed in 5 of them, whereas the other MAGE-A genes were never detected (Table 1). The levels of MAGE-A4 gene expression were assessed semiquantitatively as indicated in Table 1 and Figure 1. Among the 4 samples of HD type II that were tested, 1 (HD4) expressed gene MAGE-A4 at a level that corresponded to 10% to 30% of that found in the reference sarcoma cell line, whereas the other 3 expressed 3% to 10% (HD5) or less than 3% of that level (HD3 and HD6). The positive sample of HD type III (HD17) expressed 3% to 10% of that level. Representative results are shown in Figure 1 for patient HD11 ( ), HD5 (+), and HD3 (±). Expression of genes
MAGE-A1, -A2, -A3, -A4, -A6, and -A12 was not detected
in any of the 25 samples of normal hematopoietic cells (PBMCs,
PHA-activated peripheral T cells, bone marrow, benign lymph node, cord
blood cells) (Figure 1).
Immunohistochemical detection of the MAGE-A4 protein in HD lymph nodes The low level of expression of MAGE-A4 detected in 4 of 5 lymph nodes from HD patients could result either from a low level of transcription of the gene in most of the cells present in the sample or from a higher expression by a minority of the cells. The latter possibility was obviously relevant, considering that Reed-Sternberg cells represent usually less than 5% to 10% of the total cell population in HD lymph nodes. We therefore analyzed paraffin sections of lymph nodes from 53 HD patients with MAb 57B. This antibody recognizes cells transfected with cDNAs encoding MAGE-A1, -A2, -A3, -A4, -A6, and -A12 in tissue sections, and it stains the tumors that express gene MAGE-A4 and only these tumors, regardless of the expression of the other MAGE-A genes.28
We report the expression of MAGE-A genes in 7 of 56 lymph
nodes from lymphoma patients. Wide differences were observed according to the type of pathology. Whereas B-cell lymphomas did not express any
of the 6 MAGE-A genes that were tested, expression was found in
2 of 9 T-cell lymphomas. A higher proportion of positive samples has
been reported previously in T-cell leukemias, which expressed gene
MAGE-A1.23 A notable difference between the 2 diseases is the pathogenesis of the T-cell leukemias, namely its
frequent association with human T cell leukemia-lymphoma virus (HTLV-1) in Japan and the Philippines but not in European countries.
We are particularly grateful to Mrs. M. Swinarska and M. Van Malderen
for excellent technical assistance. We thank Dr R. Costello for helpful discussions.
Supported in part by a Swiss National Fonds grant (no.
31-57'473.99) and by grants from the Association contre le
Cancer, Brussels, Belgium; from CGER-Assurances and VIVA, Brussels,
Belgium; from the Fonds National de la Recherche Scientifique (TELEVIE grants), Brussels, Belgium; and from the Ligue Nationale contre le
Cancer, France.
Submitted October 4, 1999; accepted January 30, 2000.
Reprints: Daniel Olive, INSERM U119 and Institut Paoli
Calmettes, 27 Boulevard Leï Roure, Marseille, France; e-mail: olive{at}marseille.inserm.fr.
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.
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Top
Abstract
Introduction
in acute promyelocytic leukemia and p210
[BCR-ABL] in chronic myelocytic leukemia); proteinase 3, which is
present in leukemia cells but also in normal promyelocytes; the mucin MUC-1 in myeloma cells; the PRAME protein that appears to be
overexpressed in many acute leukemias; or viral proteins such as LMP1
and LMP2 in lymphomas associated with Epstein-Barr virus
infection.
cells from a lymph node of patient HD3. MAGE-A4
expression, analyzed with RT-PCR on the 2 cell populations, was
restricted to the CD30+ cells (data not shown).
