Abstract
Purpose: The purpose of this study was to determine the potentialof cancer testis (CT) antigens as vaccines for non-Hodgkin’s lymphomas (NHLs).
Experimental Design: Ninety-three specimens of NHLs were analyzed for their composite expression of eight CT genes (MAGE-3, MAGE-4, CT-7, HOM-MEL-40/SSX-2, SSX-1, SSX-4, HOM-TES-14/SCP-1, and HOM-TES-85). Thirty-nine of these specimens were also analyzed for their NY-ESO-1 expression.
Results: Only 1 of 7 cases of chronic lymphocytic leukemia expressed a CT gene (HOM-TES-14/SCP-1), and 10 follicular lymphomas were negative for all of the CT genes tested. In B-cell lymphomas, the most frequent expression of CT genes was observed in diffuse large-cell lymphomas (HOM-TES-14/SCP-1: 7 of 28; SSX-1: 5 of 28; CT-7: 2 of 28; and HOM-MEL-40/SSX-2 and HOM-TES-85: 1 of 28 positive cases). Only 1 of 8 Burkitt’s and 1 of 7 lymphoblastic lymphomas expressed a CT gene (CT7 and HOM-TES-14/SCP-1, respectively). A majority (9 of 15) of T- NHLs (9 peripheral T-cell lymphomas, 2 lymphoblastic T-cell lymphomas, and 4 cases of AILD) expressed HOM-TES-14/SCP-1.
Conclusions: HOM-TES-14/SCP-1, and to some degree SSX-1 and CT-7 might be candidates for lymphoma vaccine development. However, the identification of additional tumor-specific antigens with a frequent expression in lymphomas is warranted to allow for the development of widely applicable polyvalent lymphoma vaccines.
INTRODUCTION
The idiotypes of the clonotypic immunoglobulins of B-cell lymphomas (1 , 2) and the antigen receptors of T-cell lymphoma (3) , respectively, can be regarded as individual tumor-specific antigens for lymphomas. For B-cell lymphomas, idiotype vaccination has already been shown in clinical trials to be efficient (1) . However, because idiotypes have unique specificities for each individual lymphoma, they must be molecularly characterized and produced for each individual patient. This makes this approach expensive and has compromised its widespread use.
According to their expression pattern and the specificity of the immune responses they evoke, antigens expressed by human neoplasms can be classified into different groups (4) . These include the so-called “shared tumor antigens,” the differentiation antigens (including the idiotypes of B-cell lymphomas), the products of viral, mutated, differentially spliced, overexpressed, and amplified genes, as well as common autoantigens expressed by the malignant cells of a tumor. It is enigmatic that all of the so-called shared tumor antigens in humans that have been molecularly defined to date by cellular (5) and serological techniques (6) have in common their expression spectrum, which is restricted to different types of cancers and normal testis. Therefore the term CTA3 has been coined for them, and the term “cancer testis genes” for their encoding genes (7) .
The group of CT antigens includes the CTL-reactive MAGE (8) , BAGE (9) , and GAGE (10) families, as well as HOM-MEL-40/SSX-2 (11) , the other SSX family members (12) , NY-ESO-1 (7) , HOM-TES-14/SCP-1 (13) , CT7 (14) , HOM-TES-85/CT8 (15) , and CT10 (16) , all of which have been defined using SEREX, the serological identification of antigens by recombinant expression cloning (17) . The analysis of the expression pattern of the CT genes revealed that some of them are also expressed in lymphomas, for example, HOM-MEL-40, which is encoded by the SSX-2 gene (11) , and HOM-TES-14, which is encoded by the SCP-1 gene (13) . However, little is known about the composite expression of CT genes in NHLs. We now present the results of our analysis of a broad panel of CT genes in NHL. It shows that T-NHL express CT genes more frequently than B-cell lymphomas, and, hence, vaccine strategies using known CT genes are available for the majority of T-NHL, but, with the exception of diffuse large B-cell lymphomas, for only a minority of patients with B-NHL.
PATIENTS AND METHODS
Tissues and Cell Lines.
The study had been approved of by the local ethical review board (“Ethikkommission der Ärztekammer des Saarlandes”). Recombinant DNA work was done with the official permission and according to the rules of the State Government of Saarland. Tumor tissues were obtained during routine diagnostic or therapeutic procedures from University of Saarland Medical School, Homburg, Germany, and from the Institute of Hematopathology, Christian-Albrecht-Universität, Kiel, Germany. Lymphoma samples used for RT-PCR analysis were checked microscopically and with routine immunohistology. WHO lymphoma classification was used for histological diagnosis. Normal tissues were collected from autopsies of tumor-free patients.
RT-PCR.
Total cellular RNA was extracted from frozen tissue specimens using guanidium-isothiocyanate for denaturation followed by an acidic phenol extraction and isopropanol precipitation (18) . Total RNA (4 μg) was primed with a oligo(dT)18 and reverse-transcribed with Superscript II (Life Technologies, Inc., Eggenstein, Germany) according to the manufacturer’s instructions. Thus, cDNA obtained was tested for integrity by amplification of β-actin and p53 transcripts. For PCR analysis of the expression of individual CT gene transcripts 1 μg first-strand cDNA was amplified with transcript-specific oligonucleotides (10 gMol) using 2 units AmpliTaq Gold (Perkin-Elmer, Weiterstadt, Germany), 10 nmol of each deoxynucleotide triphosphate (dATP, dTTP, dCTP, and dGTP), and 1.67 mm MgCl2 in a 30-μl reaction tube. Only tumor specimens that had been assessed for cDNA integrity by amplification of both a β-actin and p53 product were investigated. Thus, of 135 available cases of fresh-frozen lymphoma tissues, 42 (31%) had to be discarded. To exclude false-positive PCR products because of small amounts of contaminating DNA in the RNA preparation, the individual primer sets were chosen for sequences that correspond to sequences located in different exons. Under the experimental conditions, DNA generated no PCR products. Each RT-PCR experiment was done in triplicate using the same polydeoxythymidylic acid-primed cDNA sample together with appropriate controls.
Amplification was performed in a TRIO-Thermoblock (Biometra, Göttingen, Germany). After 12-min activation of AmpliTaq Gold polymerase at 94°C for hot-start induction, 35 cycles of PCR were performed with 1 min at the respective annealing temperature as indicated above, 2 min at 72°C, and 1 min at 94°C with a final elongation step at 72°C for 8 min. A 15-μl aliquot of each reaction was size-fractionated on a 2% agarose gel, visualized by ethidium bromide staining, and assessed for expected size.
Immunohistology.
Formalin-fixed sections from lymphoma biopsies were deparaffinized. After extensive washing, endogenous peroxidase was blocked by treatment with 3% (v/v) H2O2. Microwave treatment was performed in citrate buffer for 5 min at 700 VA and 3 min at 350 VA. After preincubation of the slides with 1% (w/v) BSA at room temperature for 1 h, the slides were incubated for 1 h at 38°C with the monoclonal SCP-1 antibody SC554 (19) , diluted 1:10 in 1% (w/v) BSA, washed extensively, and the incubated with biotinylated rabbit antimouse immunoglobulin (DAKO) diluted in 1% (w/v) BSA for 15 min at 37°C. After another washing step, streptavidin diluted 1:300 in Tris-buffered saline was incubated for 15 min at 37°C. The reaction was developed with 3,3′-diaminobenzidine for 2–5 min at room temperature, followed by hematoxylin counterstain and embedding in entellan.
RESULTS
Study Population and Validity of the Experimental Approach.
In total, 93 tumor specimens were investigated for the expression of the following eight genes: MAGE-3, MAGE-4, HOM-MEL-40/SSX-2, SSX-1, SSX-4, HOM-TES-14/SCP-1, CT-7, HOM-TES-85, and NY-ESO-1. Because of limited amounts of cDNA available, only 39 samples were also analyzed for NY-ESO-1. There were 78 B-cell and 15 T- cell lymphomas (Table 1⇓ ⇓ ). Representative examples of RT-PCR results from a NHL are shown in Fig. 1⇓ . Intensities of PCR products were found to be heterogeneous, and some specimens yielded only faint amplicon bands. These were scored positive only, if the result could be reproduced by a repeated RNA extraction and specific PCR from the same tumor specimen. Cases with very low transcript levels, which were not reproducibly positive, were not regarded as positive.
Representative results of RT-PCR analysis for the expression of HOM-TES-14/SCP-1 in lymphomas. 1, Ly 92; 2, Ly 91; 3, Ly 98; 4, Ly 99; 5, Ly 89; 6, Ly 102; 7, Ly 125; 8, Ly 131; 9, Ly 132; 10, Ly 106; 11, Ly 132; 12, Ly 135; P, testis; N, negative control. The weak expression in Ly 132 (Lane 11) was not regarded as positive.
Expression of CT genes in NHLs
Continued
Expression of Individual CT Genes in NHLs.
As can be seen in Tables 1⇓ ⇓ and 2⇓ , NY-ESO-1 was negative in all 39 of the lymphoma samples tested. SSX-2, HOM-TES-85, MAGE-3, and MAGE-4 were expressed in only one, and SSX-4 in two cases, respectively. SSX-4 was expressed in 6 of 78 B-cell lymphomas but was absent in T-cell lymphomas. Similarly, CT-7 was found in only 4 of 78 B-cell lymphomas but not in T-cell lymphomas. The most frequently expressed CT gene was HOM-TES-14/SCP-1. It was expressed in 15 of 78 (19%) of the B-cell and 9 of 15 (60%) of the T-cell lymphomas.
Summary of CT gene expression in different histologic subtypes of NHLs
Expression of CT Genes According to Histological Subtype.
MALT and follicular lymphomas were completely negative for all of the CT genes tested, and only 1 of 7 CLL cases expressed SCP-1. The most frequent expression of CT genes in B-cell lymphomas was observed in the diffuse large cell lymphomas, where 7 of 28 cases (25%) expressed SCP-1, 5 of 28 expressed SSX-1, and 3 of 28 expressed CT-7. In contrast, expression of CT genes was rare in Burkitt’s and B-lymphoblastic lymphomas, where SSX-1, SCP-1, and HOM-TES-85 were expressed in only 1 of 15 cases.
In T-cell lymphomas, SCP-1 was expressed in the majority of the cases (9 of 15 or 60%). Apart from SCP-1, there was a rare expression of SSX-4, MAGE-3, and MAGE-4 (one of case each). All of the other CT genes were not expressed in any of the 15 T-cell lymphoma cases tested.
Coexpression of Multiple CT Genes in Lymphomas.
Expression of more than one CT gene was only observed in 4 of 28 (14%) cases with diffuse large B-cell lymphomas and in 2 of 15 (13%) of the T-cell lymphomas (Fig. 2.)⇓ . Coexpression of three CT genes occurred once among the 15 T-cell lymphomas, and coexpression of four CT genes was observed only in 1 case with diffuse large B-cell lymphoma.
Coexpression of CTAs in histological subtypes of NHL.
Demonstration of CTA Expression at the Protein Level by Immunohistology.
Because mRNA levels do not correlate strictly with protein expression, it is of interest, if mRNA-positive lymphomas express the respective antigen at the protein level. Unfortunately, only a limited number of monoclonal antibodies against the CT antigens expressed in NHL are available. Therefore, we had to restrict the analysis of protein expression to HOM-TES-14/SCP-1, the CT antigen with the most frequent expression in NHL. Of 24 HOM-TES-14/SCP-1 mRNA-positive cases, 12 were available for HOM-TES-14/SCP-1 staining. HOM-TES-14/SCP-1 protein could be demonstrated in all but 2 cases. However, the percentage of HOM-TES-14/SCP-1-positive cells within a given NHL biopsy varied considerably. A typical case is shown in Fig. 3⇓ ; in some cases the rate of positive cells was <10%.
Immunohistological detection of HOM-TES-14/SCP-1 in a diffuse large B-cell lymphoma. Positive lymphoma cells show a diffuse nuclear reactivity. Immunoperoxidase stain ×400.
DISCUSSION
To investigate a spectrum of CT genes as broad as possible despite the limited amount of cDNA that was available from each tumor, not all members of the known CT antigen families could be included in this study. Instead we selected a panel of CT genes based on known correlated expression patterns [e.g., NY-ESO-1 (7) and LAGE-1 (20)] and/or relatedness of the respective genes and gene families [e.g., the MAGE family and related genes such as CT7/MAGE-C1 (14) with CT10 (16) or DAM (21)] . Besides CT7/MAGE-C1, we chose MAGE-3 and MAGE-4 as the representatives for the MAGE group of genes, because they are the most commonly expressed of all of the MAGE genes in cancer; the same applies for SSX-1, SSX-2, and SSX-4 with respect to the SSX gene family; HOM-TES-14/SCP-1 (13) and HOM-TES-85 were included in the study panel, because their expression in lymphomas had been expected from the results of their initial expression analysis. HOM-TES-85 is a new Mr 40,000 leucine zipper protein targeting to sites of gene transcription and splicing, which was identified by screening a cDNA bank enriched for testis-specific transcripts with the serum of an allogeneic patient with seminoma (15) .
Our results show that the majority of human T-cell lymphomas and a significant proportion of diffuse large B-cell lymphomas express at least one of the shared tumor antigens, thus rendering many patients suffering from these types of NHLs eligible for trials of tumor-specific strategies. However, in other histological subtypes CT genes are rarely expressed or even absent, e.g., in follicular and mantle cell lymphomas.
The CT gene with the most frequent expression in NHLs is the gene coding for HOM-TES-14/SCP-1, which is involved in meiotic chromosome pairing (22) . Whereas its immunogenicity had originally been demonstrated in a patient with renal carcinoma (13) , antibodies against this antigen had also been demonstrated in a patient with a CTCL (23) and during the SEREX analysis of a testis-derived cDNA using the sera from patients with nodal NHL (24) , demonstrating that antibody responses against this antigen do exist in lymphoma patients. Moreover, a recently published analysis of CT gene expression in CTCLs showed that SCP-1 was expressed in cases with pleomorphic CTCL and mycosis fungoides, where all of the other CT were absent, at frequencies comparable with those observed with nodal T-cell lymphomas in this study (25) . This underlines the potential of HOM-TES-14/SCP-1 as a broadly applicable lymphoma vaccine.
From the data of our study it appears that with the exception of patients with T-cell NHL and diffuse large B-cell lymphoma only a minority of patients with other NHLs would be eligible for specific immunotherapeutic approaches with at least one CT antigen in ways similar to the ones that are being evaluated currently in malignant melanomas (26, 27, 28) , which express CT antigens at ratios between 30% and 50%. Whereas all of the patients with a lymphoma expressing a given CT antigen would be candidates for vaccine strategies using whole antigenic proteins, the percentage of patients eligible for peptide-specific vaccinations would be even lower, because it requires antigenic peptides with binding motifs restricted to specific MHC alleles. Therefore, additional antigenic CT genes must be identified for human lymphomas, especially if the development of multivalent vaccines for a majority of patients is the goal. Because the expression of a CT antigen by a tumor is a prerequisite for a strong antibody response against the respective molecule, it makes sense to exploit the expressed B-cell repertoire of lymphoma patients for the identification of novel lymphoma-associated CT genes in lymphomas. Thus, using sera from lymphoma patients should enhance the chance to identify new CT genes that have resisted discovery to date, because such a search would be biased for antibodies with reactivity to antigens with preferential expression in lymphomas. However, it is understandable that such an analysis has not been successfully performed in B-cell lymphomas because of technical problems; using the classical SEREX approach, the lymphoma-associated clonal immunoglobulin would be expressed in a cDNA library derived from a B-cell lymphoma causing false-positive reaction. Thus, alternative approaches using subtractive (e.g., immunoglobulin-deprived) cDNA libraries are necessary to achieve this goal.
Acknowledgments
We thank Claudia Schormann for excellent technical assistance.
Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 Supported by Kompetenznetz Maligne Lymphome.
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↵2 To whom requests for reprints should be addressed, at Department of Internal Medicine I, Saarland University Medical School, D-66421 Homburg/Saar, Germany. Phone: 49-68-4116-3002; Fax: 49-68-4116-3002; E-mail: michael.pfreundschuh{at}uniklinik-saarland.de
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↵3 The abbreviations used are: CTA, cancer testis antigen; CT, cancer testis; NHL, non-Hodgkin’s lymphoma; RT-PCR, reverse transcription-PCR; CTCL, cutaneous T-cell lymphoma.
- Received April 30, 2002.
- Revision received July 22, 2002.
- Accepted July 22, 2002.
References
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