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Immunology |
Departments of Human Genetics [N. R. d. S., D. R. H. d. B., E. K-B., A. G. v. K.], Tumor Immunology [R. T., M. W. J. S., G. J. A.], and Pathology [T. J. d. V., D. J. R., G. N. P. v. M.], University Hospital Nijmegen, 6500 HB Nijmegen, the Netherlands
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, when searching for melanoma tumor antigens, Türeci et al. (9) found that the SSX2 gene was expressed in melanomas and, in addition, several other malignancies. Further research disclosed that also SSX1 and two newly discovered SSX genes, SSX4 and SSX5, may be expressed ectopically in a variety of cancers (10 , 11) . In contrast, no SSX3 expression was observed in any of these malignancies. In addition, it was found that at least the SSX2 protein may act as a tumor-associated antigen (also designated as HOM-MEL-40), eliciting humoral immune responses in a subset of melanoma patients (9 , 12) . Due to the absence of expression in most normal tissues except testis and the ectopic expression in a variety of malignancies, the SSX proteins are considered as new members of the CT5 family of antigens (10) .
The SSX genes exhibit nucleotide homologies ranging from 88 to 95% and encode proteins of 188 amino acids that exhibit homologies ranging from 77 to 91%. Recent studies have shown that the SSX proteins are localized in the nucleus, exhibiting both diffuse and punctated distribution patterns (13, 14, 15) . In addition, it was found that the conserved COOH-terminus of SSX is able to repress the transcription of a reporter gene (16) . This domain is also responsible for nuclear localization, chromatin association, and colocalization with Polycomb-group nuclear bodies (17) .
In the present study, we detected variable levels of SSX RNA expression in several human melanoma and other cancer cell lines. In addition, we developed an anti-SSX monoclonal antibody suitable for SSX protein detection in paraffin-embedded tissues. Using this antibody, we detected heterogeneous SSX expression in 9 of 18 melanoma cell lines, 34 of 101 primary and metastatic melanoma cases, and 2 of 24 common nevocellular and atypical nevus cases. Normal SSX expression in the testis was found to be confined to spermatogonia. The implications of our findings for the use of SSX antigens as potential targets for immunotherapy are discussed.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the origin of which was reported previously (18
, 19) .
Eight nonmelanoma cell lines were also analyzed: UMSCC (head and neck
squamous cell carcinoma), HeLa (cervical carcinoma), A431 (cervical
epidermoid carcinoma), NT2D1 (testicular germ cell cancer), CaCo2
(colon carcinoma), A2243 (synovial sarcoma), U-4SS (sarcoma), and K562
(erythroleukemia). All cell lines were cultured and harvested as
described previously (18)
. To induce genome-wide
demethylation, cultured cells were grown in DMEM medium containing 1
µM 5-aza-2'-deoxycytidine (Sigma). After 72 h of
incubation at 37°C, the cells were processed for RNA extraction or
immunofluorescence microscopy. One hundred and twenty-five
paraffin-embedded melanocytic lesions were examined by
immunohistochemistry (listed in Table 2
). The lesions were surgically resected from patients treated at
hospital centers in Nijmegen, the Netherlands (88 lesions), Copenhagen,
Denmark (39 lesions), Würzburg, Germany (8 lesions), and
Brussels, Belgium (6 lesions). Special care was taken that all
specimens that were included in this multicenter European study were
fixed in 4% formaldehyde in PBS. Two testicular biopsies (9-year-old
and 41-year-old donors) and two normal thyroid paraffin-embedded
specimens were also examined by immunohistochemistry.
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Cloning, Restriction Analysis, and Sequencing.
SSX PCR amplification products were purified from agarose gels using
the Easy-Pure kit (Biozym) and cloned into pGEM-T vectors using the
pGEM-T Vector Systems kit (Promega) for further restriction analysis
and sequencing. Confirmation of restriction analysis results was
achieved by sequencing of the cloned PCR products using a
vector-specific primer, the ABI PRISM Dye Terminator Cycle Sequencing
Ready Reaction Kit (Perkin-Elmer), and the ABI 373A automated DNA
sequencer (Applied Biosystems).
Generation of the E3AS Anti-SSX mAb.
Mice were immunized with 10 µg of GST-SSX2 protein, produced, and
isolated from Escherichia coli, as previously reported
(13)
. During the following weeks, collected serum was
tested for reactivity on formaldehyde-fixed and paraffin-embedded 1F6
(SSX2-positive) and BLM (SSX2-negative) cells. A seropositive mouse was
boosted i.v. for 2 subsequent days with 10 µg of GST-SSX2 protein.
Two days after the last booster injection, the spleen was collected,
and the spleen cells were isolated. These cells were fused with SP2/0
myeloma cells, using PEG4000, in a ratio of 10 spleen cells:1 myeloma
cell. The hybridomas were screened by assessing the reactivity of the
various supernatants toward 1F6 and BLM cells by immunofluorescence
analyses, immunohistochemistry, and immunoblotting. Hybridomas
secreting anti-SSX antibodies were subcloned several times until
achieving monoclonality. Finally, the obtained mAbs were assessed for
staining of formaldehyde-fixed and paraffin-embedded 1F6 and BLM cells
by immunohistochemistry.
Immunoblotting Analysis.
Cultured cells were lyzed in RIPA buffer [50 mM Tris (pH
8.0), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate,
0.1% SDS, 0.1% 2-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, 4.8 µg/ml aprotinin] following
standard methods (20)
. Subsequently, equal amounts of each
cell extract were electrophoresed in SDS-polyacrylamide gels and
immunoblotted using the E3AS mAb (1:1000 dilution) and B39 polyclonal
antibody (1:3000 dilution), essentially as previously described
(13)
. As secondary antibodies, horseradish
peroxidase-coupled goat antimouse (1:1000 dilution) and horseradish
peroxidase-coupled goat antirabbit antibodies (DAKO) were used.
Detection was performed by incubating the blotted nitrocellulose
filters with enhanced chemiluminescence substrate (Amersham) and
subsequent autoradiography.
Cell Transfections and Indirect Immunofluorescence Assays.
HeLa cells were grown in DMEM medium containing 10% FCS. Different SSX
cDNAs (SSX1 to SSX4 and SSX2 deletion mutants) cloned in eukaryotic
expression vectors containing epitope tags (pCATCH and pSG8-VSV) were
transiently transfected into HeLa cells using Dosper liposomal reagent
according to the manufacturer’s instructions (Boeringher Mannheim).
Indirect immunofluorescence assays were performed as described
previously (13)
. As primary and secondary antibodies, we
used the E3AS mAb (1:10 dilution) and FITC-conjugated swine antimouse
IgG (DAKO; 1:100 dilution).
SSX Antigen Retrieval and Immunohistochemical Detection.
As a testing system for immunohistochemical staining of
paraffin-embedded pathology specimens, we embedded SSX-positive 1F6 and
SSX-negative BLM cells in gelatin. These gelatin devices were fixed in
4% formaldehyde/PBS and embedded in paraffin. Four-µm sections were
deparaffinized and treated by microwave heating for 20 min at 650 W in
0.1 mM sodium citrate buffer (pH 6.0). The slides were
adjusted to room temperature for 30 min and preincubated for 10 min in
20% normal horse serum in PBS. This solution was decanted, and the
E3AS mAb was applied as hybridoma culture supernatant for 60 min at
room temperature. We used an avidin-biotin complex-peroxidase method
for antibody detection as previously described (21)
. The
signal was enhanced with catalyzed reporter deposition, an
amplification method developed in our laboratory based on the
deposition of BT (22)
. The BT precipitate was then
visualized with peroxidase-labeled avidin and 3-amino-9-ethylcarbazole
as a substrate. The normal testis and thyroid specimens were analyzed
by immunohistochemistry using a similar protocol but without the use of
BT. In addition, these samples were stained with E3AS mAb preincubated
with GST-SSX2 or GST-SYT peptide to assess the antibody specificity.
Scoring.
For each section, the percentage of positive melanocytic cells was
estimated. Each section was assigned to one of the following percentage
categories: 0%, 1–5%, 5–25%, 25–50%, 50–75%, and 75–100%.
Positive melanocytic staining was scored when at least 1% of
melanocytic cells were stained. The scoring was performed by two
observers (N. R. d. S. and T. J. d. V.), but doubtful cases were
analyzed and judged by a panel of four observers (N. R. d. S.,
T. J. d. V., G. N. P. v. M., and D. J. R.).
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RESULTS |
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. Using primers that recognize and amplify the 3' ends of the
five known SSX genes (SSX1 to SSX5),
we observed a specific band of 324 bp in 9 of 18 melanoma cell lines
tested (Fig. 1A
; Table 1
). Eight nonmelanoma cell lines were also analyzed,
and only the U-4SS and K562 cell lines were found to be SSX-positive
(Table 1)
. In all cases, RT-PCR amplification of PBGD mRNA,
a housekeeping gene, was included as a control to ascertain the
appropriate quality and quantity of the different RNA samples (Fig. 1B)
. In nine of the melanoma cell lines, an additional
SSX RT-PCR reaction was performed using a primer set that
amplifies full-length SSX mRNAs (Fig. 1C)
.
Accordingly, the cell lines that were previously found to be
SSX-positive gave rise to a specific band of 663 bp, whereas negative
cell lines remained so, as expected (Fig. 1B)
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) indicated that more than one SSX gene was
expressed in four of nine positive melanoma cell lines. To identify the
corresponding cDNAs, the PCR fragments were cloned into plasmid vectors
and propagated in E. coli. Several of these clones were
isolated and subjected to restriction and sequencing analyses (Fig. 1C)
. By doing so, we found in three cell lines only
SSX1 (Omel2, MZ2-MEL-3.0, and U-4SS) and in three other cell
lines only SSX2 (1F6m, 518A2, and SK-mel-28) PCR products
(Table 1)
. Besides, fragments with higher molecular weight (
450 bp)
were also amplified from cell lines 518A2 and SK-mel-28 (Fig. 1A
, arrowhead). These fragments were found to
correspond to an alternatively spliced SSX2 variant
containing an additional intronic sequence of 148 bp (GenBank accession
no. AF190791). By analyzing clones derived from the remaining four
positive cell lines (1F6, 530, A375P, and A375 M), we obtained evidence
for the expression of the SSX4 and SSX5 genes
recently identified by Güre et al. (10)
in three of them. SSX4 expression was detected in the 530
and A375 M cell lines, whereas SSX5 expression was detected
only in the A375P cell line (Table 1)
. The SSX1 and
SSX2 genes were both expressed in these four cell lines. It
should be noted that no SSX3 gene expression was found in
any of the melanoma cell lines tested.
Generation and Characterization of an Anti-SSX mAb.
For further analysis at the protein level, we set out to generate a
mouse mAb against SSX proteins. Several hybridomas were produced, and
because we were particularly interested in developing an antibody able
to detect protein from paraffin-embedded tissues, each hybridoma was
assessed for its ability to detect SSX expression in paraffin-embedded
1F6 cells. BLM cells, which were SSX-negative by RT-PCR, were used as a
negative control. By doing so, we identified one hybridoma (E3AS) that
was able to specifically detect SSX in 1F6 but not in BLM cells (Fig. 4, a-c).
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and tested them for reactivity by immunofluorescence analysis. Absence
of the most NH2-terminal 110 amino acids (as in
standard SYT-SSX fusions; FLAG-SYT-SSX2) led to loss of E3AS mAb
staining. Conversely, COOH-terminal deletions up to amino acid 81 did
not abrogate E3AS staining. Because absence of the 24 most
NH2-terminal amino acids did not prevent E3AS
recognition, the epitope of this mAb maps between amino acids 25 and 80
(Fig. 2)
.
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. The expression was heterogeneous, and only a fraction of
spermatogonia was stained. Likewise, in immature testis, SSX expression
was found to occur in a portion of spermatogonial cells, the
predominant cell type present (data not shown). Neither interstitial
cells nor Sertoli cells were stained by the E3AS mAb. Attesting the
specificity of the mAb E3AS, we found that preincubation with the
immunizing antigen (GST-SSX2) abolished nuclear staining of
spermatogenic cells. In contrast, preincubation with an unrelated
protein (GST-SYT) had no effect on E3AS staining (data not shown).
Surprisingly, none of the thyroid tissue sections examined revealed SSX
staining.
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).
Immunoblotting using the B39 antibody revealed specific bands of 29 kDa
in 1F6, 1F6m, and 518A2 cell lysates, as well as in a positive control
(COS-1+SSX2; Fig. 5A
). Less intense bands of similar weight
could be discerned in the immunoblotted SK-mel-28, A375P, and A375 M
cell lysates. Identical results were obtained using the E3AS mAb (not
shown). Immunofluorescence analysis using the E3AS mAb disclosed
nuclear stained cells in 9 of 13 analyzed cell lines. However, the
percentage of positive cells varied markedly between cell lines. In
three of them (1F6, 1F6m, and 518A2), the majority of cells were
positive (>80%), whereas five other cell lines (Omel2, 530, A375P,
A375 M, and MZ2-MEL-3.0) showed very low numbers of positive cells
(<2%). The SK-mel-28 cell line showed an intermediate percentage of
positive cells (25%; Table 1
).
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, no SSX staining was found
in the remaining benign human melanocytic lesions (Fig. 4l)
.
Within the malignant lesions, the frequency of E3AS positivity ranged
from 24% in locoregional metastases to 40% in primary melanomas
(Table 2)
. Among all lesions studied, SSX expression was very
heterogeneous, with the majority of the cases exhibiting low numbers of
positive cells (<25%; Table 2
). Large SSX-negative areas were
observed in several tumors. Four main staining patterns could be
distinguished in both primary and metastatic melanomas: (a)
widespread, with >75% of positive cells (Fig. 4d)
;
(b) focal, with positive cells clustered in a restricted
area from the tumor (Fig. 4e)
; (c) scattered,
with a low percentage of positive cells present in several areas of the
tumor (Fig. 4f)
; and (d) isolated, with few
positive cells localized in largely negative areas (Fig. 4g)
. Apart from the typically observed nuclear staining,
some cases showed aberrant patterns. In one primary tumor, cytoplasmic
staining of mitotic cells was observed next to nuclear staining in
other cells (Fig. 4h)
. In seven melanoma lesions (two
primary melanomas and five metastases), tumor cells showed cytoplasmic
or membrane staining, either exclusively or in conjunction with the
typical nuclear staining (Fig. 4i)
. Also, one lymph node
metastasis exhibited SSX staining in the cytoplasm of fibroblast-like
cells adjacent to tumor cells (Fig. 4j)
.
Because 32 patients had both primary and metastatic tumors, we
investigated whether the status of SSX expression varied in the
different lesions from each patient (Table 3)
. Fourteen patients developed both primary and metastatic SSX-negative
lesions, whereas in six patients, all lesions showed SSX nuclear
positivity. Six patients had SSX-negative primary melanomas and at
least one SSX-positive metastasis, whereas six other patients had
SSX-positive primary tumors and negative metastases. Furthermore, no
correlation was found between SSX expression and Clark staging, Breslow
tumor thickness, or age of the patients.
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. The effect of DAC treatment on SSX expression
was also assessed at the protein level for BLM cells.
Immunofluorescence microscopy revealed that DAC treatment resulted in
SSX nuclear staining in about 9–13% of the cultured cells (Fig. 5C)
. In contrast, no cells were stained in the absence of
DAC. Furthermore, incubation with higher concentrations of DAC (10
µM) did not increase the percentage of
SSX-positive BLM cells (7%; not shown). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of 18 melanoma cell lines studied by RT-PCR, 9 were found to express at
least one SSX gene, SSX1 and SSX2
being most frequently expressed (Table 1)
. The levels of SSX
expression were variable, as also found by Güre et al.
(10)
in their panel of melanoma cell lines. In three pairs
of melanoma cell lines derived from the same patient (A375P/A375 M,
1F6/1F6m, and MV1/MV3), the overall presence or absence of
SSX expression was concordant. However, the SSX
family members were different in each pair, suggesting that the
expression of certain SSX genes might have been switched on
or off after cell line establishment. Using a newly developed mAb
(E3AS) that recognizes the NH2 terminus (amino
acids 25–80) of SSX2, SSX3, and SSX4 (but not SSX1) proteins, we
searched for expression in 13 of 18 melanoma cell lines by
immunoblotting and immunofluorescence. In contrast to the former
method, which revealed SSX bands in only six cell lines,
immunofluorescence analysis allowed the detection of SSX protein in all
cell lines that were found to be SSX-positive by RT-PCR. However, the
percentage of SSX-positive cells varied considerably among these cell
lines (Table 1)
. A low percentage of SSX-positive cells was observed in
two cell lines (530 and MZ2-MEL-3.0) that showed strong SSX
expression by RT-PCR. This discrepancy is likely due to the fact that
the E3AS mAb does not detect SSX1 expression. Altogether, these results
indicate that the different levels of SSX mRNA detected by RT-PCR are
not due to differential intensities of transcriptional activity but
rather to cell-to-cell heterogeneity.
The E3AS mAb was designed and found to be suitable for the detection of SSX protein in paraffin-embedded tissues. Using this antibody, we analyzed 125 cases of benign and malignant melanocytic lesions by immunohistochemistry. Heterogeneous SSX expression was found in 40% of primary melanomas analyzed. This percentage is similar to what has been observed by other investigators using RT-PCR (43% [11]), but it may be underestimated because SSX1 expression is not detected by this antibody. Yet, this underestimation may not be of major significance because others found that only 1 of 37 (3%) melanomas tested expressed SSX1 alone (11) . The heterogeneity of SSX expression in the melanocytic lesions was striking, ranging from widespread to scarce. This finding parallels our observations in the melanoma cell lines and, therefore, indicates that SSX-expressing cells do not undergo positive or negative selection during cell line establishment.
Thirty-two patients from our series developed both primary and
metastatic melanomas (Table 3)
, allowing the assessment of whether SSX
expression could be related to tumor progression. Because six patients
had SSX-positive primary melanomas and SSX-negative metastases, we
conclude that expression of these proteins does not confer a clonal
selective advantage to melanoma cells in vivo. This is in
contrast with the reported association between MAGE
expression and tumor progression and increased aggressiveness
(19
, 23)
.
The SSX proteins are members of the still growing family of CT antigens. All CT antigens (e.g., MAGE, BAGE, GAGE, NY-ESO-1/LAGE-1, SCP-1, CT7, and SSX) share common characteristics: (a) normal expression in the testis only, (b) ectopic expression in malignancies of various histological origins, (c) existence of several family members, and (d) localization on the X chromosome (10 , 24 , 25) . However, not all CT antigens fully comply with these rules. For example, several MAGE genes are also expressed in the placenta (26) , SSX1 and SSX2 RNAs are detected in the thyroid (4 , 6) , and the SCP-1 gene is localized on chromosome region 1p12-p13 (27) . SSX expression at the protein level was detected in the testis but not in two normal thyroid specimens examined by immunohistochemistry (this report). Additional RT-PCR analyses were not performed on these samples due to lack of fresh and/or frozen material. However, our results combined with those of others that did find SSX expression in the thyroid by RT-PCR (4 , 6) suggest that this expression may be too low to be detected at the protein level. Alternatively, the discrepancy may be explained by a bias in sample biopsies.
Immunohistochemical detection of SSX proteins in the testes of two donors revealed nuclear expression in spermatogonia, both from mature and immature testes. Several studies have shown that spermatogenic cells undergo profound changes in methylation patterns (28, 29, 30) . In several cases, it was observed that hypomethylation of certain promoter sequences occurs at the early stages of spermatogenesis (i.e., in spermatogonia), thereby being linked to the activation of transcription (29 , 31 , 32) . SSX expression in spermatogonia suggests that also these genes may be activated after demethylation of their promoter sequences. Our finding that DAC incubation induces SSX expression in otherwise non- or low-expressing cells further supports this hypothesis. A similar phenomenon has been observed for MAGE-1, a gene that is also primarily expressed in spermatogonia (33) and is activated by DAC treatment (19 , 34) .
The testis is an immune privileged organ because spermatogenic cells do
not express HLA class I and II antigens at the cell surface (35
, 36)
. Concomitantly, the testis has a so-called blood-testis
barrier in the seminiferous tubuli generated by the Sertoli cells.
Because ectopic expression of CT antigens may thus lead to an
autologous cellular and/or humoral immune response (12
, 37)
, these antigens are presently being used as targets for the
development of cancer immunotherapy protocols. Several clinical trials
have been set up with relative success seeking immunization against CT
or melanocyte-specific antigens (e.g., gp100, tyrosinase,
and MART-1/Melan-A) in metastatic melanoma patients
(38, 39, 40, 41)
. As CT antigens, the SSX proteins may also be
immunogenic when ectopically expressed and, therefore, be suitable as
targets for immunotherapy. Previous studies have reported the presence
of anti-SSX antibodies in the sera of melanoma patients (9
, 12)
. In the present study, we found that the proportion of
SSX-expressing cells varied considerably among the different samples
tested, thereby raising the question as to whether all
SSX-positive tumors would be equally suitable for immunotherapy.
Heterogeneous expression in melanomas has also been reported for the
MAGE-1 protein (42
, 43) , but the MAGE proteins tend to be
uniformly expressed in multiple melanoma lesions from the same patient
(44)
. In contrast, we found that 12 of our patients
developed both SSX-negative and -positive lesions (Table 3)
, suggesting
that, in such cases, anti-SSX immunotherapeutic approaches would not be
equally effective for all these lesions. Also, melanoma cases with a
low percentage of SSX-expressing cells may be less suitable for
immunotherapy unless a so-called bystander effect occurs,
i.e., if the SSX-targeted action of CTLs would create a
cytokine environment, triggering an inflammatory response that would
kill neighboring tumor cells (45)
.
Because DAC induces SSX expression in vitro, it would be of interest to study the effectiveness of DAC as a coadjuvant to SSX vaccination in cases with low levels of SSX expression. Previously, DAC has been shown to inhibit tumor growth and induce differentiation (46 , 47) , and it has been successfully administered to leukemia patients as a therapeutic agent (48) . Furthermore, the recent observation that DAC also up-regulates the expression of HLA class I antigens and costimulatory molecules (e.g., ICAM-1 and LFA-3) in cultured melanoma cells (49) suggests that this agent may enhance the immunogenicity of tumor cells and, therefore, may serve as an important coadjuvant in immunotherapeutic protocols targeting SSX and/or other CT antigens for the treatment of melanoma.
In conclusion, the generation of a mAb (E3AS) specifically directed against SSX proteins allowed the detection of these proteins in a series of primary and metastatic melanoma lesions. The use of this antibody revealed a high level of heterogeneity among the different cases studied. This finding stresses the importance of assessing SSX expression at the protein level in melanoma cases that may be selected for immunotherapeutic trials. However, because the E3AS mAb does not recognize SSX1 protein, RT-PCR amplification remains useful to complement immunohistochemistry to evaluate more accurately the status of SSX expression in tumors. Despite the relatively low levels of SSX expression in melanomas when compared with other antigens (e.g., MAGE, gp100, and tyrosinase), we maintain that melanoma immunotherapy targeted to SSX should be contemplated, particularly in those cases with high levels of SSX protein expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants NUKC95–912 and NUKC96–1351
from the Dutch Cancer Society (Koningin Wilhelmina Fonds) and Grant
PRAXIS XXI/BD/3232/94 from the Fundação para a
Ciência e Tecnologia (Lisbon, Portugal; to N. R. d. S.). 
2 To whom requests for reprints should be
addressed, at University Hospital Nijmegen, P. O. Box 9101, 6500
HB Nijmegen, the Netherlands. Phone: 31-24-3618847; Fax:
31-24-3540488.
3 These authors contributed equally to this
work.
4 Present address: Department of Cell Biology and
Histology, Academical Medical Center, P. O. Box 22700, 1100 DE
Amsterdam, the Netherlands.
5 The abbreviations used are: CT, cancer/testis;
BT, biotinylated tyramine; DAC, 5-aza-2'-deoxycytidine; GST,
glutathione-S-transferase; RT, reverse
transcription; mAb, monoclonal antibody.
Received 10/15/99. Accepted 1/20/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 F. Ebstein, C. Sapede, P.-J. Royer, M. Marcq, C. Ligeza-Poisson, I. Barbieux, L. Cellerin, G. Dabouis, and M. Gregoire Cytotoxic T Cell Responses against Mesothelioma by Apoptotic Cell-pulsed Dendritic Cells Am. J. Respir. Crit. Care Med., June 15, 2004; 169(12): 1322 - 1330. [Abstract] [Full Text] [PDF]
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 T. Takeuchi, H. H. Q. Heng, C. J. Ye, S.-B. Liang, J. Iwata, H. Sonobe, and Y. Ohtsuki Down-Regulation of a Novel Actin-Binding Molecule, Skeletrophin, in Malignant Melanoma Am. J. Pathol., October 1, 2003; 163(4): 1395 - 1404. [Abstract] [Full Text] [PDF]
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M. Ayyoub, D. Rimoldi, P. Guillaume, P. Romero, J.-C. Cerottini, D. Valmori, and D. Speiser Tumor-reactive, SSX-2-specific CD8+ T Cells Are Selectively Expanded during Immune Responses to Antigen-expressing Tumors in Melanoma Patients Cancer Res., September 1, 2003; 63(17): 5601 - 5606. [Abstract] [Full Text] [PDF]
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 M. Maio, S. Coral, L. Sigalotti, R. Elisei, C. Romei, G. Rossi, E. Cortini, F. Colizzi, G. Fenzi, M. Altomonte, et al. Analysis of Cancer/Testis Antigens in Sporadic Medullary Thyroid Carcinoma: Expression and Humoral Response to NY-ESO-1 J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 748 - 754. [Abstract] [Full Text] [PDF]
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Y. Xie, B. Skytting, G. Nilsson, A. Gasbarri, K. Haslam, A. Bartolazzi, B. Brodin, N. Mandahl, and O. Larsson SYT-SSX Is Critical for Cyclin D1 Expression in Synovial Sarcoma Cells: A Gain of Function of the t(X;18)(p11.2;q11.2) Translocation Cancer Res., July 1, 2002; 62(13): 3861 - 3867. [Abstract] [Full Text] [PDF]
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 V. Anne Westbrook, A. B. Diekman, S. Naaby-Hansen, S. A. Coonrod, K. L. Klotz, T. S. Thomas, E. J. Norton, C. J. Flickinger, and J. C. Herr Differential Nuclear Localization of the Cancer/Testis-Associated Protein, SPAN-X/CTp11, in Transfected Cells and in 50% of Human Spermatozoa Biol Reprod, January 1, 2001; 64(1): 345 - 358. [Abstract] [Full Text]
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