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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 2 748-754
Copyright © 2003 by The Endocrine Society

Analysis of Cancer/Testis Antigens in Sporadic Medullary Thyroid Carcinoma: Expression and Humoral Response to NY-ESO-1

Michele Maio, Sandra Coral, Luca Sigalotti, Rossella Elisei, Cristina Romei, Guido Rossi, Enzo Cortini, Francesca Colizzi, Gianfranco Fenzi, Maresa Altomonte, Aldo Pinchera and Mario Vitale

Cancer Bioimmunotherapy Unit (M.M., S.C., L.S., E.C., F.C., M.A.), Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, 33081 Aviano, Italy; Dipartimento di Biologia e Patologia Cellulare e Molecolare (G.R., M.V.), Università Federico II, 80131 Naples, Italy; Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, Università Federico II, Naples, Italy (G.F.); and Istituto di Endocrinologia ed Oncologia Sperimentale "G. Salvatore" (G.R.), Consiglio Nazionale delle Ricerche, Istituto di Endocrinologia (R.E., C.R., A.P.), 56124 Pisa, Italy

Address all correspondence and requests for reprints to: Mario Vitale, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Via S. Pansini 5, 80131 Napoli, Italy. E-mail: mavitale{at}unina.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cancer/testis antigens (CTA) are tumor-associated antigens expressed during ontogenesis, in a number of solid tumors but not in normal tissues except testis. Most of these CTA are highly immunogenic, eliciting a humoral and cellular response in the patients with advanced cancer, and are useful for tumor-specific immunotherapy. Medullary thyroid carcinoma (MTC) is a neoplasm derived from the parafollicular cells of the thyroid and occurs in either a sporadic or a familial form. In the present study, we examined by RT-PCR the expression of a number of genes encoding CTA in 23 surgical samples of sporadic MTC. Among the 11 cDNA antigens examined, RAGE, MAGE-4, and GAGE 1–2, were not expressed in any of the tissues. SSX 2 was present only in one tissue, whereas BAGE, GAGE 1–6, MAGE-1, MAGE-2, MAGE-3, and SSX 1–5 were detected in two to five samples. NY-ESO-1 cDNA was the most frequent, being detected in 15 of 23 examined samples (65.2%). Six (26.1%) tissues did not express any CTA-specific mRNA, whereas 10 tumors expressed only one gene (43.5%), 3 (21.4%) expressed 2 genes, and 4 displayed a broad CTA gene expression. NY-ESO-1 expression in primary MTC tissues significantly correlated with tumor recurrence. The presence of specific anti-NY-ESO-1 antibodies was searched in the sera of MTC-affected patients examined by ELISA using recombinant NY-ESO-1 protein. A humoral response against this CTA was detected in 6 of 11 NY-ESO-1 expressing patients (54.5%), and in 1 of 6 patients with NY-ESO-1-negative tumor. No anti-NY-ESO-1 antibodies were detected in healthy subjects (n = 17). The presence of anti-NY-ESO-1 antibodies was searched also in the sera of MTC affected patients whose tissues were not available for CTA analysis. Anti-NY-ESO-1 antibodies were present in 15 of 42 sera (35.7%), demonstrating that MTC is a neoplasm frequently associated with humoral immune response to NY-ESO-1. Serological survey may be useful as a way to identify patients with humoral immune response to NY-ESO-1 that provide a new attractive target for vaccine-based immunotherapy of MTC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEDULLARY THYROID CARCINOMA (MTC) is a neoplasm derived from the parafollicular or C-cell of the thyroid. MTC accounts for 5–10% of thyroid malignancies, has a peak incidence in the fifth and sixth decades, is slightly more common in women, and occurs in either a sporadic or a familial form (1, 2, 3). The sporadic form is more frequent and slightly more aggressive than the familial variety that represents 21–25% of all MTC. Sporadic MTC typically appears as a unilateral and unifocal enlarging thyroid nodule confined to the thyroid gland at the time of presentation. Although it is an aggressive cancer, MTC remains a differentiated tumor and presents endocrinologic symptoms due to large calcitonin (CT) secretion. Surgical resection is currently the only successful therapy for both sporadic and familial form of MTC (4). Prognosis is determined by the form of MTC, by the stage of disease at presentation and by the age of the patient. The presence of distant metastasis dramatically worsen the prognosis to less than 15% of patients surviving 10 yr. Chemotherapeutic agents such as adriamycin, 5-fluoruracil, bleomycin, cis-platinum or methotrexate, alone or in combination, failed to significantly increase survival (5, 6, 7, 8). It is hoped that the development of new adjuvant therapies will significantly improve the clinical course and prognosis of MTC in the future.

Tumor-associated antigens (TAA) are molecules exclusively or mainly expressed in tumors. Some of these TAA are expressed in melanoma cells as well as in normal melanocytes and in many tumors including bladder, breast, and lung carcinomas (9, 10, 11, 12, 13), whereas they are not present in normal adult tissues, except for the testis. TAA can be distinguished into the following categories according to their pattern of expression: 1) differentiation-specific antigens, expressed in normal and neoplastic melanocytic cells, such as tyrosinase, Melan-A/MART-1, gp100/Pmel17, and TRP-1/gp75; 2) cancer/testis antigens (CTA), expressed in melanomas and other solid tumors but not in normal tissues except testis, such as the MAGE, GAGE, and SSX gene families, BAGE and NY-ESO-1; 3) antigens derived from point mutations of ubiquitously expressed proteins, such as MUM-1, ß-catenin, HLA-A2, CDK4, and caspase 8; 4) antigens overexpressed in malignancies of different histology but also present in several normal tissues, such as PRAME (14). A number of TAA have been identified as useful therapeutic targets for active immunization of melanoma patients, and promising results have been obtained (15, 16, 17, 18). Most of these CTA are highly immunogenic, eliciting spontaneous immune responses either cellular (19, 20) or associated with a strong humoral component (21) in the patients with advanced cancer. For this reason, patients with ascertained immunologic response are potential candidates for tumor-specific immunotherapy directed against TAA. Among known TAA, CTA represent optimal therapeutic targets due to their confined expression in normal tissues and to their demonstrated in vivo immunogenicity. Thus, to evaluate whether vaccination against CTA-derived peptides may represent a potential therapeutic strategy for the treatment of patients affected by MTC, in the present study, we examined the expression of different CTA in surgical samples of sporadic MTC and their humoral immunogenicity, demonstrating the frequent expression of NY-ESO-1 gene. Furthermore, we analyzed by ELISA the presence of anti-NY-ESO-1 antibodies in the sera of patients, demonstrating that MTC is the neoplasm most frequently associated with spontaneous humoral immune response to this antigen. The results of this preliminary study suggest that CTA-based immunotherapeutic approaches may represent a therapeutic strategy that can be pursued in MTC patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients, tissues, and sera

Twenty-three patients (10 males and 13 females, age at diagnosis ranging 21–71 yr) were treated with total thyroidectomy after MTC diagnosis based upon clinical data (neck mass, elevated serum CT) and fine needle aspiration biopsy. All clinical data including age, gender, tumor node metastasis stage, and outcome were obtained from the clinical-pathologic records. The presence of disease after surgery was evaluated by pentagastrin test and whole-body computed tomography scan, and patients were classified in the groups "disease free" or "tumor recurrence." Fresh tumor tissues were immediately frozen in liquid nitrogen after surgical resection and stored at -80 C until RNA extraction. Diagnosis was confirmed by histology. Sera were collected from MTC patients at diagnosis prior of any therapy. Patient’s sera and normal human sera (NHS) from healthy volunteers were stored at -20 C until use. All patients and normal controls gave informed consent for participation in this study.

CT measurement

Serum CT was measured with a commercial kit (a solid phase two-site immunoradiometric assay-IRMA-ELISA-human CT; Cis, Gif-sur-Yvette, France) with a sensitivity of 14 pg/ml. The upper limit of normal range in the laboratory was 20 pg/ml, with most normal controls (86%) having undetectable serum CT levels (<14 pg/ml). Pentagastrin-stimulated serum CT assay was performed as previously described (22).

Analysis of RET mutations by PCR assay

Genomic DNA was purified from fresh tissues by overnight proteinase K digestion at 55 C, followed by a phenol/chloroform extraction and ethanol precipitation. When a RET mutation was found on tumoral DNA, genomic DNA was extracted and analyzed from peripheral blood lymphocytes using the QIAMP blood kit (QIAGEN, Hilden, Germany), and DNA was kept in Tris EDTA at -20 C.

Genomic DNA was amplified twice for each exon by PCR using a PTC-100 instrument (MJ Research, Inc., Watertown, MA) and specific primers for exons 10, 11, 13, 14, 15, and 16 (23). One hundred nanograms of genomic DNA were mixed in final volume of 100 µl with 10 mM Tris-HCl; 50 mM KCl; 0.1% Triton X-100; 1.5 mM MgCl2; 100 pmol of each primer for exons 11; and 200 pmol of each primer for exons 13, 14, and 15, 250 µM deoxynucleotide triphosphates; and 5 U Taq polymerase. Thirty-five cycles of denaturation (94 C for 5 min), annealing (60 C for exons 13 and 15, 65 C for exons 10, 11, and 14, 53 C for exon 16 for 1 min), and extension (72 C for 2 min) were performed. Following the amplification, 5 µl of PCR product were electrophoresed in a 2% agarose gel to verify the quality of the amplification. Sequence analysis was performed on 95 µl of the PCR product previously purified with a commercial kit (Concert Rapid PCR Purification System, Life Technologies, Inc., Milan, Italy) and sequenced using an automated system employing dye fluorescent terminators (ABI Prism 310; Perkin-Elmer, Foster City, CA). RET mutation analysis was extended to peripheral white blood cells. No germline mutations were found.

Analysis of CTA expression by RT-PCR assay

Total RNA was extracted from tissue samples of MTC using the TRIzol reagent (Life Technologies, Inc.), according to the manufacturer’s instructions. Reverse transcription was performed on 2 µg total RNA, and PCRs were performed on 5 µl cDNA using 50 pmol of each sense and antisense primer as previously described (24). Oligonucleotide primer sequences and gene-specific PCR amplification programs used have been previously defined for MAGE-1, -2, -3, -4 (25), NY-ESO-1 (19), GAGE 1–2 (10), GAGE 1–6 (10), SSX 1–5 (26), SSX 2 (27), BAGE (9), RAGE-1 (28), tyrosinase and Melan-A/MART-1 (29). The integrity of RNA and oligo(deoxythymidine)-synthesized cDNA and the nature of the tissue specimens were confirmed by amplification of all cDNA samples with ß-actin- and CT-specific primers, respectively, as previously described (24). Ten microliters of each RT-PCR sample were run on a 2% agarose gel and visualized by ethidium bromide staining.

Prokaryotic expression cloning of NY-ESO-1

To produce full-length NY-ESO-1 recombinant protein, the coding sequence for the protein was PCR amplified from cDNA of the NY-ESO-1-positive HT-1080 fibrosarcoma cell line, and cloned into BamHI/KpnI sites of pQE30, a plasmid vector containing histidine tags (QIAGEN, Chatsworth, CA). PCR primers for NY-ESO-1 amplification were ESO1-S 5'-CATCACGGATCCATGCAGGCCGAAGGCCGG-3' and ESO1-AS 5'-ACCCGGGGTACCGCGCCTCTGCCCTGAGGG-3'. After transformation into Escherichia coli strain M15[pREP4], positive transformants were confirmed to contain the appropriate insert by restriction mapping and DNA sequencing. Recombinant NY-ESO-1 protein was then produced by isopropyl ß-D-thiogalactoside induction and purified by Ni2+ affinity chromatography, following procedures recommended by the manufacturer (QIAGEN). Concentration of the purified protein was determined by colorimetric protein quantification assay (Bio-Rad Laboratories, Inc., Hercules, CA).

ELISA

One hundred microliters per well of 0.25 µg/ml recombinant NY-ESO-1 protein in coating buffer [15 mM Na2CO3; 30 mM NaHCO3 (pH 9.6)] were adsorbed to 96-well U-bottom microtiter flexible plates (Becton Dickinson and Co., Oxnard, CA) overnight at 4 C. Plates were washed with PBS and blocked 2 h at room temperature with 200 µl/well of PBS containing 2% BSA. After washing, 100 µl/well of serum dilutions were added and incubated for 2 h at room temperature. Plates were washed, and 100 µl/well of diluted (1:10000) secondary antibody (peroxidase-conjugated goat antihuman IgG and antihuman IgM, ImmunoResearch Lab. Inc., West Grove, PA) were added and incubated for 1 h at room temperature. After washing, plates were developed, and OD was read at 405 nm. Sera were tested over a range of serial 2-fold dilutions from 1:100 to 1:409600. A positive reaction is defined as an OD value of 1:800 diluted serum that exceeds the mean OD value of sera from normal donors (n = 17) by 3 SD values.

Statistical analysis

The sample tumors that either expressed or did not express each CTA gene were compared. Statistical analysis was performed by using the Fisher’ exact test method. The level of significance was set at P less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of CTA expression by RT-PCR

A characteristic of MTC is to remain a differentiated tumor and to produce CT, although it is a malignant and aggressive cancer. To confirm the specificity of the PCR products, the nature of the tissue specimens analyzed, and to ensure that the RNA was not degraded, a PCR assay with primers specific for actin, CT, tyrosinase, and MART-1 cDNA was carried out for each specimen. CT and actin mRNA expression was detected in all tissue samples, whereas no expression of the melanocytic-differentiation antigens tyrosinase and MART-1 was detected, confirming that all samples were well preserved, suitable to be analyzed by PCR and of thyroid medullary origin. Twenty-three MTC tissue samples were analyzed for their constitutive expression of CTA. Results are reported in Table 1Go and summarized in Fig. 1Go. RT-PCR analysis revealed a heterogeneous expression of CTA mRNA within distinct MTC lesions studied (Fig. 2Go). Of the 14 cDNA antigens examined, RAGE, MAGE-4, PRAME, MART-1, GAGE 1–2, and Tyrosinase were not expressed in any of the tissues, whereas SSX-2 was present only in one tissue and BAGE, GAGE 1–6, MAGE-1, MAGE-2, MAGE-3, and SSX-1–5 were detected in two to five samples (8.7–21.7%). NY-ESO-1 cDNA was that most expressed, being detected in 15 out 23 examined samples (65.2%).


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  		Table 1. RT-PCR analysis of CTA in MTC tissue samples

 


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  		Figure 1. CTA mRNA expression in tissue samples from 23 MTC patients. Total RNA was extracted from tissue samples of 23 patients affected by MTC. RT-PCR analysis was performed using CTA-specific primer pairs, and PCR products were then separated on a 2% agarose gel. Data report the number of patients with neoplastic lesions expressing investigated CTA.

 


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  		Figure 2. Representative RT-PCR analysis of NY-ESO-1 expression in tumor samples from 13 MTC patients. Total RNA was extracted from tissue samples. RT-PCR analysis was performed with NY-ESO-1-specific primer pairs, and PCR products were then separated on a 2% agarose gel. One hundred-base pair marker was run in the flanking lane of gel.

 
The expression of at least one CTA was observed in 17 (73.9%) of 23 MTC studied, whereas 6 (26.1%) of them did not express any cDNA antigen. Ten tumors expressed only one gene, 3 (21.4%) expressed 2 genes, and 4 displayed a broad CTA gene expression.

Correlation among CTA gene expression, clinicopathologic features, and RET mutations

The expression of CTA mRNA determined by RT-PCR and the clinical characteristics of patients, including age, gender, stage of disease, serum CT, RET mutations, and outcome, were examined (Table 2Go). Expression of CTA did not correlate with sex, age at diagnosis, nor with RET mutations. However, NY-ESO-1 antigen expression correlated with disease outcome. Patients were followed for 4–7 yr and divided in two groups based upon outcome. Patients with negative pentagastrin-stimulated serum CT assay and whole-body computed tomography scan were classified in the group "disease free" otherwise in the "tumor recurrence." The presence of tumor recurrence was found in 11 of 15 NY-ESO-1 positive patients and in 2 of 8 NY-ESO-1 negative patients. The association between NY-ESO-1 antigen expression and tumor recurrence was statistically significant by Fisher’s exact test (Table 3Go).


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  		Table 2. Clinical characteristics of the patients

 

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  		Table 3. Correlation between NY-ESO-1 antigen expression and disease outcome after 5–7 yr of follow-up

 
Detection of circulating antibodies to NY-ESO-1 in MTC patients

Serial dilutions of 17 NHS and sera from 42 unselected MTC-affected patients were analyzed for the presence of circulating anti-NY-ESO-1 antibodies before surgery. Fifteen patients (35.7%) had anti-NY-ESO-1 antibodies (Fig. 3Go). Fresh-frozen tumor specimens and serum samples were available from 17 patients. Six of 11 patients with NY-ESO-1-positive tumors had anti-NY-ESO-1 antibodies (Table 4Go, Fig. 4Go). No detectable anti NY-ESO-1 antibodies were present in 5 patients with NY-ESO-1-positive tumors. Notably, high titer anti-NY-ESO-1 antibodies were also detected in patient 16, who has an NY-ESO-1-negative tumor. No anti-NY-ESO-1 antibodies were detected in the remaining 5 patients with NY-ESO-1-negative tumors, or in 17 healthy subjects. A possible correlation between the presence of circulating anti-NY-ESO-1 antibodies and disease outcome was searched in this series of MTC patients. Although Ab anti-NY-ESO-1 were more frequently found in patients with recurred disease, the correlation between immune response and disease outcome was not significant by two-sided Fisher’s exact test (P > 0.5921). However, the group of patients analyzed included several subjects studied up to 3 yr after surgery, a follow-up too short to establish a valid correlation between immune response and disease outcome.



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  		Figure 3. Survey of anti-NY-ESO-1 antibodies in sera of MTC patients. ELISA reactivity of sera from 42 unselected MTC patients was tested against NY-ESO-1 recombinant protein. A positive reaction is defined as an OD value of a 1:800 diluted serum that exceeds the mean OD value of NHS by 3 SD values (dashed line). Data represent OD values of 1:800 diluted sera from patients with anti-NY-ESO-1 antibodies (filled triangles) and from patients with no anti-NY-ESO-1 antibodies (open triangles).

 

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  		Table 4. Correlation between NY-ESO-1 mRNA expression anti-NY-ESO-1 antibodies in sera of 17 MTC affected patients

 


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  		Figure 4. Representative results of anti-NY-ESO-1 antibodies in sera from MTC patients. ELISA reactivity of sera from four selected MTC patients with NY-ESO-1-positive (no. 11, dots; no. 13, triangles) or NY-ESO-1-negative (no. 2, squares; no. 16, crosses) tissue samples was tested against NY-ESO-1 recombinant protein. Data represent OD values obtained by one representative experiment out of three.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The MAGE, BAGE, GAGE, and NY-ESO-1 genes have been shown to be expressed in several cancers such as melanoma, breast, gastric, and hepatic tumors. In this study, we present a novel finding demonstrating the presence of different CTA in tissue samples of sporadic MTC. The tumors examined displayed an etherogeneous expression of CTA. Seven of 23 MTC expressed more than one CTA gene: 1 CTA was expressed in 10 tissues, and 6 did not express any antigen. The relationship between disease progression and the expression of these CTA has been investigated in large series of melanomas and bladder carcinomas (25, 30). Expression of MAGE-1, MAGE-2, and MAGE-3 was significantly increased in larger melanomas and MAGE-1, MAGE-3, GAGE, and BAGE were significantly higher in metastatic lesions than in the primary lesions. A correlation between clinicopathological characteristics and CTA expression was demonstrated also in bladder tumors. MAGE, BAGE, and GAGE genes were more frequently expressed in more invasive bladder carcinomas than in those less invasive. Our study demonstrate a significant correlation between NY-ESO-1 expression and disease outcome. NY-ESO-1 positivity in primary MTC tissues significantly correlated with tumor recurrence. To further investigate these aspects and establish stronger relationships between CTA expression and characteristics of the patients or disease outcome, a prospective study with a larger series of samples is needed. The expression of the CTA under investigation was not studied in normal parafollicular cells. Specific antibodies required for immunohistochemistry are available only for few CTA (6). Thus, as parafollicular cells are rare and sparse within the thyroid gland, in situ hybridization with specific nucleotide probes is the only technique that could be used to study CTA gene expression at a single-cell level. However, the identification of new molecules associated to MTC, useful in the diagnosis of thyroid nodules, was not the aim of this study as CT production, measured in the serum and in situ by immunohistochemistry are sufficient to ensure a correct diagnosis.

The main goal of the present study was to identify the immunogenic CTA that are expressed in MTC and to evaluate the presence of a spontaneous humoral immune response against selected CTA in patients affected by MTC. The main finding of our study is the frequent expression of NY-ESO-1 gene (65.2%). The expression of this antigen in MTC was higher than in melanoma (34%), hepatoma (28%), in breast (30%), prostate (25%), and ovarian (25%) cancer, whereas normal cells (except for testis and ovary) and most tumors (colon, gastric, renal and pancreatic carcinomas, glioma, leiomyosarcoma, seminoma, and lymphoma) are completely negative. The restricted expression of NY-ESO-1 in testis, an immunologically privileged anatomical site, likely explains the potential for cancer-related immune recognition of this antigen. Indeed, both humoral and cellular immune reactivity has been frequently observed in patients with melanoma and ovarian cancer (10%) and with an even lower frequency in lung and breast cancer (21). Noteworthy, NY-ESO-1 is the most immunogenic antigen defined to date. Indeed, this CTA elicits a strong humoral and cellular immune response in a high proportion of patients with NY-ESO-1-expressing tumors (20, 21). The availability of a screening system to analyze the presence of anti-NY-ESO-1 antibodies allowed us to investigate the NY-ESO-1 antibody status in 42 MTC unselected patients. Results showed that anti-NY-ESO-1 antibodies were found in 35.7% of patients, a frequency comparable with melanoma and ovarian cancer and higher than in breast and lung cancer (16). These data and the presence of anti-NY-ESO-1 antibodies in 6 of 11 patients with NY-ESO-1-positive tumors demonstrate that NY-ESO-1 expressed by MTC is immunogenic.

The presence of anti-NY-ESO-1 antibodies in a patient with an NY-ESO-1-negative tumor is rather surprising. A possible explanation is the presence of an unknown NY-ESO-1 expressing metastasis or tumor heterogeneity not represented in the small tissue fragment analyzed by RT-PCR. It can also be speculated that this specific tumor was originally NY-ESO-1-positive and the elicited anti-NY-ESO-1 antibody response lead to the clearance of the NY-ESO-1-positive tumor cells, leaving NY-ESO-1-negative tumor cells unaffected. This hypothesis is supported by previous observations obtained in melanoma patients vaccinated with MART-1 and tyrosinase immunogenic peptides (32). The recent evidence that NY-ESO-1-specific CD8+ T cell responses are detected in more than 90% of anti-NY-ESO-1 antibody-positive melanoma patients (20) suggests a possible T cell response also in MTC patients. A correlation study between NY-ESO-1 antibody response and clinical features and disease outcome in a large series of MTC patients might help to clarify the role of immune response in this cancer and provide useful information on the prognosis.

Although preliminary, our data strongly suggest that CTA-based immunotherapy might have a crucial role in a prospective therapeutic strategy for the treatment of MTC patients. Particularly, active immunization with NY-ESO-1 peptides may represent a useful therapeutic option for the treatment of MTC patients, according to preliminary promising results obtained by the vaccination of patients affected by melanoma and other tumors (16, 33). Stimulation of dendritic cells with specific antigens is a promising method for the induction of antitumor immunity in patients with malignant diseases (34, 35, 36). CT, Carcino embryonic antigen, and tumor cell lysates have been used to stimulate dendritic cells of MTC affected patients for induction of antitumor immunity (37, 38). The use of a highly immunogenic antigen such as NY-ESO-1 might induce a more specific and stronger immunologic response against MTC cells.


   Acknowledgments
 


   Footnotes
 
This work has been partly supported by Ministero dell’Università e della Ricerca Scientifica, Programa di Rilevante Interesse Nazionale (to G.R.), by Associazione Italiana per la Ricerca sul Cancro (to M.M.), by Consiglio Nazionale delle Ricerche, Comitato Scienze Biologiche e Mediche (to M.V.), and by Ministero della Salute, Progetto Ricerca Finalizzata (to M.M.).

Abbreviations: CT, Calcitonin; CTA, cancer/testis antigens; MTC, medullary thyroid carcinoma; NHS, normal human sera; TAA, tumor-associated antigens.

Received May 29, 2002.

Accepted November 7, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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