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Cancer Immun
Cancer Immunity
1424-9634Academy of Cancer Immunology
090909
Article
Real-time PCR analysis of genes encoding tumor
antigens in esophageal tumors and a cancer vaccine
Brian T. Weinert1, Kausilia K. Krishnadath2, Francesca Milano2, Ayako W. Pedersen1, Mogens H. Claesson1 and Mai-Britt Zocca1
1DanDrit Biotech A/S, Copenhagen, Denmark
2Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, Netherlands
Communicated by: LJ Old
(9 October 2009) Vol. 9, p. 9
Submitted: 6 July 2009. Accepted: 14 September 2009.
Copyright © 2009 by Mai-Britt Zocca
Tumor antigens are the primary target of therapeutic cancer
vaccines. We set out to define and compare the expression pattern of
tumor antigen genes in esophagus carcinoma biopsies and in an
allogeneic tumor lysate-based cancer vaccine, MelCancerVac®. Cells
used for vaccine production were treated with the DNA
methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) to
determine whether this treatment could improve the profile of tumor
antigen genes expressed in these cells. In addition, the presence of
MAGE-A tumor antigen protein was evaluated in the purified tumor
cell lysate used in the production of the vaccine. Quantitative PCR
was used to assay 74 tumor antigen genes in patients with squamous
cell carcinoma of the esophagus. 81% (13/16) of tumors expressed
more than five cancer/testis (CT) antigens. A total of 96 genes were
assayed in the tumor cell clone (DDM1.7) used to make tumor cell
lysate for vaccine preparation. Gene expression in DDM1.7 cells was
compared with three normal tissues; 16 tumor antigen genes were
induced more than ten-fold relative to normal tissues. Treatment
with 5-aza-CdR induced expression of an additional 15 tumor
antigens to a total of 31. MAGE-A protein was detected in cell lysate
by Western blot at an estimated concentration of 0.2 µg/ml or 0.01%
of the total protein.
Keywords: human, esophageal cancer, MelCancerVac®, tumor
antigen, 5-aza-2'-deoxycytidine, PCR
Introduction
Cancer of the esophagus is the sixth leading cause of cancer
mortality worldwide. Since 1975 the incidence of esophageal
adenocarcinoma has increased 6-fold, with a corresponding
7-fold increase in mortality [reviewed in (1, 2)]. Overall, cancers
of the esophagus and stomach have limited therapeutic options
and 5-year survival is less than 20%. Esophageal tumors are
usually treated by surgical resection with poor outcomes.
Furthermore, recent studies using radiation or radiation/
chemotherapy combination therapy have shown limited clinical
benefit. The lack of good therapeutic options for esophageal
cancer indicates that immunotherapy could play a role in
treating this disease by inducing anti-tumor immunity (1).
Immunotherapy is a particularly attractive treatment option due
to the common expression of CT antigens in esophageal tumors
(3-5), and the lack of serious toxicity or adverse side effects
resulting from dendritic cell-based therapies (6-8).
Cancer immunotherapy aims to activate the immune system
to recognize tumor antigens, proteins that are specifically
expressed by tumor cells and most importantly, displayed on the
cell surface as MHC-peptide antigen complexes. Tumor
antigens are defined in two main groups: (i) Tumor specific
antigens (TSAs) are generally unique to tumor cells and result
from mutation of normal genes or from expression of oncogenic
viral proteins. (ii) Tumor associated antigens (TAAs) are normal
human proteins that are abnormally overexpressed in tumor
cells. An important class of TAAs is the cancer/testis (CT) class
of genes; also known as cancer germline genes, these genes are
only expressed in tumor cells and in testis (6, 9). Many CT class
genes are regulated by DNA methylation and are therefore
activated by chemicals that inhibit DNA methyltransferases,
such as 5-aza-2'-deoxycytidine (5-aza-CdR) (10-13). The
restricted expression of CT genes enables the immune system to
recognize antigenic peptides derived from these gene products.
Cytotoxic T lymphocytes have been found that recognize CT
antigens in cancer patients in the absence of immunotherapy,
indicating that the immune system can recognize and target
such antigens (14, 15). Many clinical vaccine studies have
targeted CT genes; however, only a few of these studies have
shown a significant effect on disease progression in cancer
patients (16, 17), with the recent exception of GlaxoSmithKline's
MAGE-A3 antigen-specific cancer immunotherapeutic (ASCI)
[data presented at the 2008 American Society of Clinical
Oncology (ASCO) Annual Meeting in Chicago - abstracts
9065(1), 9045(2) and 7501(3) and reviewed in (18)]. Many
clinical studies use only a single CT gene product or antigenic
peptide to induce anti-tumor immunity. It may be that a small
number of antigenic peptides are not sufficient to induce tumor
rejection, while immunotherapy that targets the greatest
number of possible TAAs is more likely to effectively target
tumor cells.
MelCancerVac® is a therapeutic cancer vaccine that is based on
loading patient-derived dendritic cells with an allogeneic tumor
cell lysate. A tumor cell lysate is used as the source of tumor
antigen in order to provide the most diverse and abundant
source of tumor antigens. By using a cell lysate there is no
selection for any particular tumor antigen or a particular
recipient HLA type. Thus, this vaccine approach aims to induce
immunity against the broadest possible range of tumor antigens,
including both known and unknown antigens. The specific
melanoma cell clone used to produce tumor cell lysate for
vaccine production is DDM1.7.
In the present study we have assayed for the expression of a
large number of tumor antigen genes in a total of 16 biopsies
from squamous cell tumors of the esophagus. In addition, we
examined the molecular expression of tumor antigen genes in
DDM1.7 cells with and without treatment with 5-aza-CdR, in
three normal tissues, in an esophageal tumor biopsy, and in
normal human testis. The protein expression of MAGE-A was
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evaluated in whole cells and purified cell lysate by Western blot.
Our goal is to define those tumor antigens whose expression can
be monitored by reverse transcription-quantitative polymerase
chain reaction (RT-QPCR) and to use this approach to identify
patient tumors that have a similar expression of tumor antigens
as the DDM1.7 cells used to generate tumor vaccine. This
approach can be used to identify a patient group for treatment
with a cancer vaccine, as in this study, or to select individual
patients for treatment based on overlapping expression of tumor
antigen genes in tumors and in a vaccine.
Results
Tumor antigen gene expression in esophageal tumor biopsies
Expression of 74 tumor antigen genes (64 of which are CT type
tumor antigen genes) in esophageal tumor biopsies from 16
patients (Table 1) was assayed by RT-QPCR. Biopsies were taken
together with matched normal tissue biopsies, in order to
compare gene expression in a patient tumor to the neighboring
normal tissue. However, not all normal tissue biopsies yielded
usable RNA. Therefore, to enable consistent comparison of
tumor samples to normal tissue, the average relative expression
values for normal tissue biopsies from 8 patients was
determined and compared with the relative expression level in
purified esophagus RNA purchased from Ambion
(Supplementary Figure 1). Gene expression was similar in all
normal esophagus tissue samples; we therefore used the average
relative expression in normal tissue biopsies from 8 patients as a
baseline value to compare with tumor samples. In the reactions
where no gene product was detected in normal tissue (such as
for MAGE-A1), the value of 10-6 relative to GAPDH expression
was set as the detection limit and induction of expression was
calculated relative to this value. The 10-6 cutoff value was chosen
for two reasons: (i) It is approximately equal to the minimal
expression detected in our system (see Table 2 and
Supplementary Figure 1). (ii) Dilution experiments showed this
to be the detection limit for several genes that are not detected in
normal tissue (data not shown). Furthermore, in order to
identify only those genes that show robust gene induction we
ignored changes in gene expression that were less than 10-fold in
magnitude.
The results of gene expression analysis in esophageal tumor
biopsies are shown in Figure 1. Expression of CT tumor antigens
is frequent in squamous cell carcinoma of the esophagus, with
81% (13/16) of biopsies expressing more than five CT tumor
antigens, while 63% (10/16) express more than 10 CT tumor
antigens (Figure 1b). The most frequently expressed antigens
were the MAGE-A and MAGE-B genes, as well as CSAG,
IL13R
α
2, BRDT, and HCA661. Tumors that express MAGE-A3
expressed high numbers of tumor antigens in general (64%
expressed 20 or more tumor antigens; only one biopsy expressed
less than 10 tumor antigens, P05, which expressed 9), indicating
that this gene may be a good marker for CT expression overall.
We used a pan-MAGE-B reaction to detect expression of
multiple MAGE-B genes (MAGE-B). Detection of MAGE-B by
this reaction correlated well with detection of individual
MAGE-B family genes (10/13), indicating that this reaction may
be used in pre-screening for MAGE-B expression. However, it
should be noted that the MAGE-B primer pair is not particularly
specific; it is able to amplify many MAGE-B family members by
allowing amplification even in the presence of mismatched base
pairs and may amplify related MAGE genes, such as MAGE-A8.
Table 1
Patients included in this study.
A total of 9 tumor antigen genes were not detected in any of the
tumor biopsies while a total of 26 tumor antigen genes were
detected in fewer than 25% of the biopsies. Non-CT type tumor
antigen genes were rarely induced in tumor biopsies (p53,
TPBG, BCL-XL, MCL1, BCL2
α
, livin/BIRC7 and survivin/
BIRC5), with the exception of the hTERT gene, which was
modestly induced in 25% of tumor biopsies.
Tumor antigen gene expression in DDM1.7 cells
A larger number of QPCR reactions was used to detect tumor
antigen gene expression in the cells (DDM1.7) used to make
tumor cell lysate for MelCancerVac®. The purpose of assaying
tumor antigen gene expression in DDM1.7 melanoma cells is to
identify highly expressed tumor antigen genes. Total RNA
isolated from normal skin, lung, and esophagus tissues was used
to determine the baseline expression of tumor antigen genes in
normal tissue. Human testis RNA was used as a positive control
for gene expression since many of the genes we assayed are CT
type TAAs, which are known to have robust expression in testis.
This also provides a means to compare the degree of CT gene
expression in a tumor tissue to the normal gene expression level
in testis. The esophagus tumor biopsy from patient 4 (P04,
Figure 1) was used as an additional positive control since this
sample showed robust tumor antigen gene expression.
The result of the QPCR analysis is shown in Table 2 using
symbols to indicate gene expression relative to GAPDH. The
actual relative expression values are given in Supplementary
Table 1. For many genes the results were consistent with CT type
tumor antigens, i.e. expression was very low or absent in normal
tissues and elevated in tumor tissues and testis (see MAGE-A1).
However, some CT genes had surprisingly high expression in
normal tissue, in contrast to their classification as CT-specific.
Similarly, some genes were detected at a low level in only one of
the normal tissue samples, indicating partially restricted
expression.
Since we were interested in determining which genes may be
significantly expressed in DDM1.7 cells, we set a cutoff value for
gene expression at 10-fold higher relative expression than in
normal tissues. Using this cutoff we identify 16 genes as being
elevated in DDM1.7 cells (MAGE-A1, MAGE-A2, MAGE-A3,
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Table 2
Analysis of tumor antigen gene expression by QPCR.
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Figure 1
Tumor antigen gene expression in esophagus tumor biopsies. (a) Gene expression in tumor samples is compared to the expression in normal esophagus tissue. Gene
expression changes less than 10-fold in magnitu de are not shown. Frequency of gen e expression in the 16 tumor biops ies is shown on the right hand side of the figure. (b)
Frequency of overa ll tumor antigen expression in t he 16 tumor biopsies is shown.
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Figure 2
Gene expression induced by 5- aza-2 -deoxycytidine in DDM1.7 cells. The graph shows the gene expression in DDM1.7 cells treated with 5-aza-CdR for 72 hours rela-
tive to DDM1.7 cells. Error bars i ndicate standard deviation bet ween reaction duplicates.
MAGE-A6, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2,
MAGE-B6, GAGE, BAGE, SAGE, CAGE, CSAG, IL13-R
α
2, and
TPTE). Treatment of cells with the DNA methyltransferase
inhibitor 5-aza-CdR nearly doubles the number of genes with
elevated expression to 31 genes (MAGE-A1, MAGE-A2,
MAGE-A3, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12,
MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B6, MAGE-B18,
MAGE-C1, MAGE-C2, GAGE, BAGE, SAGE, PAGE, CAGE,
LAGE2, SSX, CSAG, IL13-R
α
2, TPTE, BORIS, DPPA2, MMA,
HOM-TES-85, CTAGE, NY-SAR-35, and FTHL). Furthermore,
a direct comparison of gene expression in DDM1.7 cells to
DDM1.7 cells treated with 5-aza-CdR shows that the expression
of 27 genes is elevated by treatment with 5-aza-CdR by a factor
of more than 10 (Figure 2). Significantly, only CT type tumor
antigens were induced by treatment with 5-aza-CdR while other
genes such as hTERT, p53, and the inhibitor of apoptosis class
genes (BCL-XL, MCL1, BCL2a, and survivin) were largely
unaffected. In addition, CT genes that were already significantly
elevated in DDM1.7 cells (such as MAGE-A1) were not further
induced by treatment with 5-aza-CdR, suggesting that these
genes may be hypomethylated in DDM1.7 cells before
treatment with 5-aza-CdR.
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Figure 3
Gene expression relative to normal esophagus tissue (ESO) in an esophageal tumor (P04), DDM1.7 cells with and without 5-aza-CdR treatment. Only the genes
that were expressed >10-fold higher than normal esophagus tissue in at least one of the three samples are shown. The data is taken from Table 1. Note the similarity in
gene expression between 5-azaCdR treated cells and the esophageal tumor.
Tumor antigen gene expression in the esophageal tumor
biopsy (patient 4) is more extensive than in DDM1.7 cells but is
comparable to the expression seen in DDM1.7 cells treated with
5-aza-CdR (Figure 3). Only the genes that have a greater than
10-fold higher expression level than the tissue-matched normal
esophagus sample (patient 4) are compared in Figure 3. Note
that 37 genes have significantly higher expression in the
esophageal tumor biopsy sample; of these 37 genes, 95% (35/37)
are also highly expressed in DDM1.7 cells treated with 5-aza-
CdR and 60% (22/37) are genes that were significantly induced
by 5-aza-CdR in DDM1.7 cells.
Assaying MAGE-A protein expression in DDM1.7 cell lysate
While the expression of tumor antigen genes is likely to
indicate the presence of tumor antigen protein in cells or tissues,
it remains important to verify and quantify the amount of tumor
antigen protein that is present. Our vaccine product,
MelCancerVac®, is based on loading patient-derived dendritic
cells with a purified cell lysate made from DDM1.7 cells. In
order for vaccine-induced T cells to effectively target a
particular tumor antigen, the tumor antigen protein must be
present in both DDM1.7 cells and in the purified cell lysate.
MAGE-A protein was assayed in DDM1.7 cells and purified
cell lysate by Western blot with the 6C1 monoclonal antibody
(Figure 4a). MAGE-A protein was detected in both purified
DDM1.7 cell lysate (melanoma cell lysate, MCL) and in whole
cell lysate made by lysis of DDM1.7 cells in
radioimmunoprecipitation assay (RIPA) buffer. Including
protease inhibitor and/or proteasome inhibitor in the RIPA lysis
buffer had no effect on protein stability (data not shown). Three
independently produced batches of MCL were assayed with
similar results (data not shown). A standard curve of purified
MAGE-A fusion protein enables us to estimate the abundance of
MAGE-A protein in MCL (Figure 4b). Western blots were
developed with a fluorescent scanner, allowing direct
quantification of signal intensity using ImageQuant software.
These data show that the MCL batch analyzed in Figure 4
contains approximately 1 ng MAGE-A/5 µl MCL or 200 ng/ml.
Since the MCL batch contains a total of approximately 2.2 mg/
ml of protein, MAGE-A represents approximately 0.01% of the
total protein present. Attempts to detect additional CT antigens
with antibodies directed against MAGE-C1, SSX, IL-13Rα2, and
LAGE1/NY-ESO-1 all failed due to insufficient signal or high
background (data not shown).
Discussion
In this study we show that squamous cell carcinomas of the
esophagus frequently express CT type tumor antigen genes. We
further demonstrate that DDM1.7 cells, used to provide tumor
antigen for the production of MelCancerVac®, express many of
the same tumor antigen genes found in squamous cell
carcinoma of the esophagus. Treatment of DDM1.7 cells with
the DNA methyltransferase inhibitor 5-aza-CdR induces further
expression of CT type tumor antigen genes, thereby increasing
the number antigens that may be targeted by a cancer vaccine.
MAGE-A tumor antigen was detected in purified lysate
prepared from DDM1.7 cells, indicating that this tumor cell
lysate contains this tumor antigen protein.
In order to identify tumor antigen genes expressed in
squamous cell carcinomas of the esophagus, and in DDM1.7
melanoma cells, we assayed a large number of tumor antigen
genes simultaneously. Gene expression (mRNA) typically
indicates whether or not the gene product (protein) is present in
the cells or tissue being analyzed. However, gene expression
alone cannot be used to specify the presence or absence of tumor
antigen. Protein expression may be regulated at the level of
protein stability rather than gene transcription, and tumor
antigens may arise by abnormal processing of normally
expressed proteins (19). In addition, at least one ubiquitously
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Figure 4
MAGE-A protein is present in DDM1.7 cell lysate. (a) Western blot using the pan-MAGE-A monoclonal antibody 6C1 (reacts with MAGE-A1, -A2, -A3, -A4, -A6,
-A10, and -A12). Samples analyzed: “ng MAGE-A” indicates the amount of MAGE-A fusion protein loaded in each lane, MCL corresponds to melanoma cell lysate,
DDM1.7 RIPA refers to whole cell lysate made with RIPA buffer, SK-MEL-28 is a positive control cell lysate, CCD1064-SK is a negative control cell lysate. (b) Graph
showing the MAGE-A standard cur ve made by measuring the fluorescent intensity of MAGE-A fusion protein in the Western blot shown above.
expressed human protein has been shown to become antigenic
in cancer patients (20). Analysis of tumor antigen gene
expression by QPCR is rapid, inexpensive, and easy. However,
this approach can only be used to assay tumor antigens that are
transcriptionally activated in tumors. Therefore, the main
reason for performing this work is to identify the tumor antigen
genes that can be monitored by RT-QPCR in patient tumor
samples, in order to identify patients that may benefit from
treatment with MelCancerVac® or other immune therapies and
as a first step in designing patient-tailored immune therapy. In
addition, by measuring a large number of genes simultaneously
we can compare the expression levels of different tumor antigen
genes directly, an insight that is missing from the many studies
that examine the expression of one or a few tumor antigen genes
at a time.
We found that a fairly large number of CT tumor antigen genes
were expressed to a significant degree in normal tissue,
apparently contradicting the initial studies that classified these
genes as CT restricted. We do not have the space here to review
the literature for each CT gene individually; however, two
studies in particular also found expression of CT genes in
normal tissues and are consistent with the results presented
here. A recent study examined CT gene expression using
existing gene expression data found in a multitude of gene
expression databases and libraries (21). This study identified
three groups of CT genes: Testis-restricted, testis/brain-
restricted, and testis-selective. Genes that were identified as
testis-selective had gene expression in some normal tissues that
was typically less than expression in testis. Our results largely
agree with the results presented in this study. However, we
detected a very low level of expression in some of our normal
tissue samples for genes that are classified as testis-restricted.
This may result from the higher sensitivity of the RT-QPCR
assay used in our study or from inadequate coverage in the gene
expression libraries used in (21). Similarly, Scanlan et al. (9)
surveyed CT gene expression by RT-PCR and similarly found
expression in a number of normal tissues. Such results indicate
that the initial classification of CT genes may be unreliable and
that a comprehensive study of CT gene expression is needed to
allow for better selection of antigens for immune therapy. The
data presented here and in (9, 21) are a step in the right
direction.
Analysis of tumor antigen gene expression in squamous cell
carcinoma biopsies shows that CT antigen expression occurs in
a large fraction of this patient group (81%). The frequency of
MAGE-A gene expression was similar to that observed in
previous studies using immunohistochemistry (3, 5). Activation
of the MAGE-A3 gene is always accompanied by activation of
additional CT genes, making MAGE-A3 a good marker for CT
gene activation overall. As seen in other studies, CT gene
activation is clustered; if a single CT gene is activated, it is likely
that additional genes are also activated (22). This observation
supports the idea that CT gene activation results from a loss of
DNA methylation. The gene expression profile of IL13R
α
2 and
CSAG differs from the "MAGE" genes in that increased
expression is not always linked with MAGE-A3 expression.
IL13R
α
2 and CSAG are ubiquitously expressed in normal tissues
[(9) and our study] and do not appear to be significantly
induced by 5-aza-CdR, perhaps indicating that activation of
these genes occurs by a different mechanism than activation of
the "MAGE" genes. Overall these results indicate that it may be
possible to treat squamous cell carcinoma of the esophagus with
immune therapy, such as vaccination or adoptive T cell transfer
targeting CT antigens. In support of this idea, dendritic cells
loaded with total RNA from esophagus tumor cells are able to
activate T cell-dependent killing of primary esophagus tumor
cell cultures as compared to normal cells (23). Tumor antigen
expression in DDM1.7 cells treated with 5-aza-CdR overlaps
with 95% of the tumor antigen genes expressed in the tumor
biopsy from patient 4. This indicates that MelCancerVac®
contains many tumor antigens that are shared with squamous
cell tumors of the esophagus. In addition, these results underline
the necessity to screen patients for CT antigen expression before
attempting immune therapy that targets CT antigens, as some
patients do not express CT antigens at all.
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We find that treatment with 5-aza-CdR induces the activation
of many CT tumor antigen genes. This simple treatment doubles
the number of known tumor antigens expressed by DDM1.7 and
broadens the range of antigens targeted by MelCancerVac®. The
comparison presented here does not identify all of the genes that
may be induced by 5-aza-CdR, as some genes are already
expressed at a high level in DDM1.7 and may not be further
induced by treatment with 5-aza-CdR. For example, a number
of studies have shown that MAGE-A1 is induced by 5-aza-CdR
(24-26). However, in DDM1.7 MAGE-A1 expression is elevated
before treatment with 5-aza-CdR and is not further induced by
treatment with 5-aza-CdR. In fact, a large number of CT genes
are induced by treatment with 5-aza-CdR (MAGE-A, LAGE-1,
LAGE-2, SSX-2, CAGE, GAGE, HAGE, and PAGE- 5 [(10-12) and
reviewed in (13)]). In contrast, some CT genes were not induced
by treatment with 5-aza-CdR in our study (MAGE-B4, SCP1,
OY-TES-1, PLU-1, TPTE, ADAM2, SP17, MMA, D40, LDHC,
SGY-1, FATE , LIP1, and SPO11). Of these genes, SP17 and
LDHC have previously been shown to be regulated by DNA
methylation and induced by treatment with 5-aza-CdR (27, 28).
Therefore, treatment with 5-aza-CdR may not induce gene
expression in an equivalent manner in different tissues or cell
lines.
DDM1.7 cells treated with 5-aza-CdR have overlapping
expression with 95% of the genes found to be overexpressed in
the esophageal tumor biopsy sample with the highest degree of
tumor antigen expression (patient 4). Therefore, a high degree of
CT tumor antigen expression is mirrored by cells treated with
5-aza-CdR. This observation suggests that DNA demethylation
is responsible for much of the CT tumor antigen expression
observed in some squamous cell tumors of the esophagus.
Several studies have shown that promoter methylation regulates
the expression of CT genes (29-32). Since some tumor biopsy
samples have little or no tumor antigen gene expression
whatsoever (Figure 1), these data further suggest that loss of
DNA methylation only occurs in some tumors, resulting in the
expression of CT type tumor antigens, while other tumors are
unaffected. It is therefore important to treat only those patients
that express CT tumor antigens with therapies designed to target
CT antigens. DNA methyltransferase inhibitors, such as 5-aza-
CdR and related compounds, have been approved for use as
therapeutic agents targeting myelodysplastic syndrome and
myelogenous leukemia (33, 34). These compounds inhibit
tumor cell growth by reactivating genes that have been silenced
by DNA methylation in cancer cells. However, treatment of
patients with 5-aza-CdR may also facilitate immunotherapy by
inducing CT antigen expression in patient tumors, an
observation that has also been made by others (35).
As mentioned above, the expression of a tumor antigen gene is
not sufficient in itself to indicate the presence of tumor antigen
protein in cells or tissues. The tumor cell lysate component of
MelCancerVac® is prepared by lysing DDM1.7 cells and
removing insoluble cellular debris by several rounds of
centrifugation and sterile ultrafiltration. Therefore, it is possible
that protein may be lost during the lysis and purification steps.
Unfortunately, only a few quality antibodies are commercially
available for assaying tumor antigen expression by Western blot.
In this study we detected MAGE-A protein using the pan-
MAGE-A 6C1 monoclonal antibody (the antibody reacts with
MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12). We detect
MAGE-A in the purified tumor cell lysate, indicating that
MAGE-A proteins in the lysate can be loaded onto patient
dendritic cells. Quantification of proteins by Western blots is
often imperfect; however, we estimated the amount of MAGE-A
protein present by comparing Western blot signal intensity to a
standard curve made with purified MAGE-A fusion protein.
Although the relative quantity of MAGE-A is a small fraction of
the total protein (0.01%), this amount may be sufficient to
induce MAGE-A specific CTL responses in patients vaccinated
with lysate-loaded dendritic cells.
In conclusion, in this study we identify tumor antigen genes
that may be monitored for expression by RT-QPCR. While not
suitable for all tumor antigens, this approach provides a rapid
and inexpensive method to screen patients for tumor antigen
gene expression to identify patient groups suitable for
immunotherapy or to select individual patients for
immunotherapy, based on overlapping expression of tumor
antigen genes in a patient tumor and in a cancer vaccine.
Treatment with the DNA methyltransferase inhibitor 5-aza-CdR
induces CT type tumor antigen gene expression and may be
used to improve tumor antigen gene expression in tumor cell
lysate-based cancer vaccines. Tumor antigen protein abundance
should be analyzed in lysate-based therapies in order to
determine if the amount of tumor antigen protein present is
sufficient to induce an immune response.
Abbreviations
5-aza-CdR, 5-aza-2'-deoxycytidine; CT, cancer/testis; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; MCL, melanoma
cell lysate; QPCR, quantitative polymerase chain reaction; RT-
QPCR, reverse transcription-QPCR
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Materials and methods
Patients
Between July 2007 and September 2008, twenty patients with a
suspicion of esophageal squamous cell carcinoma were referred
to the Department of Gastroenterology and Hepatology of the
Academic Medical Center (AMC) for investigation by upper
gastrointestinal endoscopy. The study was approved by the
Medical Ethics Committee of the AMC. All patients gave
informed consent with written permission for the study. Patients
underwent endoscopic ultrasonography for tumor and local
node stage classification and biopsies were taken for
confirmation of the diagnosis. All the procedures were
performed by one endoscopist/investigator. During the
procedure, 4 biopsies were taken for the study: 2 from normal
tissue and 2 from the tumor. At least one matching biopsy, taken
from the same spot of the study biopsies, was collected for
histopathological diagnosis. This so-called 'correlating biopsy'
served as a control for the diagnosis and presence of cancerous
tissue in the study biopsies. Out of the twenty patients biopsied,
sixteen were included. Two patients did not show cancerous
tissue in the correlating biopsy, one had adenocarcinoma of the
esophagus, and one biopsy had an insufficient yield of RNA to
perform the analysis. Of the sixteen patients four were females,
mean age was 61 (range 53 to 75). At endoscopy, two patients
had cancer reaching the muscularis propriae (T2), the other
fourteen had cancers beyond the muscularis propriae and outer
esophageal wall but without invasion of the neighboring organs
(T3). All patients had lymph node metastasis (N1). Details are
given in Table 1.
Preparation of RNA and cDNA
Total human RNA was purchased from Stratagene (MVP Total
RNA, Human Skin #540031; the distributor in Denmark is A.H.
Diagnostics, Aarhus, Denmark) and Ambion (Human Lung
Total RNA #AM7968, Human Esophagus Total RNA #6842;
Austin TX, USA). RNA was isolated from cell lines and
esophagus tissue biopsies using the Qiagen RNeasy mini kit
(Qiagen AB, Solna, Sweden) with DNAse treatment in solution.
Total RNAs purchased from Stratagene and Ambion were also
treated with DNase and re-purified using the Qiagen RNeasy
mini kit. Cell culture samples (approximately 2 x 106 cells) were
lysed in Qiagen lysis buffer (RLT) and stored at -80°C until
purification was performed. Esophagus tissue biopsies (20 mg -
30 mg) were placed in RNAlater (Ambion) reagent overnight at
4°C then stored at -80°C until RNA was purified. Tissue biopsies
were frozen in liquid nitrogen and homogenized with a mini-
pestle in a microfuge tube while frozen and, when thawed, in the
presence of Qiagen RLT lysis buffer. Homogenized tissue lysate
was then treated by centrifuging with a QIAshredder column to
remove tissue debris and the sample further homogenized
before proceeding with the Qiagen RNeasy purification. Total
RNA was quantified using a UV spectrophotometer or by using
Ribogreen reagent (Invitrogen, Taastrup, Denmark) with a RNA
standard curve. Complementary DNA (cDNA) was synthesized
from total RNA using Superscript III reverse transcriptase
(Invitrogen) as per the manufacturer's protocol. Each 25 µl
reaction contained 1 µg total RNA, 400 nM oligo dT24, and
400 nM random hexamer. Reverse transcription reactions were
diluted with 100 µl H2O and stored at -20°C.
Quantitative PCR
Quantitative PCR was performed using a Stratagene Mx3000P
instrument and the data analyzed with MxPro software.
Individual reactions contained 10 µl Brilliant SYBR Green
Master Mix (Stratagene), 2 µl cDNA (equivalent to 16 ng total
RNA), oligonucleotides at a final concentration of 500 nM and
H2O for a total volume of 20 µl. The thermo cycles were as
follows: 1 cycle of 10 min at 95°C; 40 cycles of 30 s at 95°C, 60 s
at 56°C, 30 s at 72°C, and 1 dissociation cycle of 30 s at 95°C, 30 s
at 55°C, slow ramp to 95°C. All QPCR runs included no reverse
transcriptase control reactions and a dissociation analysis of the
final products in order to confirm the formation of specific PCR
products from mRNA. PCR products amplified from testis RNA
were analyzed by agarose gel electrophoresis to confirm that the
PCR products were the correct length (data not shown). QPCR
reactions were performed in duplicate for each sample. RT-
QPCR was used to detect the presence of 96 tumor antigen genes
using 74 reactions that amplify sequences from 153 transcripts.
Transcripts amplified and PCR primers used can be found in
Supplementary Table 2. Relative gene expression was
determined by comparing gene expression to glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) expression. In each QPCR
reaction run a GAPDH standard curve was made by 1,000-fold
serial dilution (10x at each point) and the PCR efficiency was
determined. The PCR efficiency was then used to calculate the
abundance of cDNA for each gene relative to GAPDH. We
included QPCR analysis of an additional two housekeeping
genes, TATA box-binding protein (TBP) and
hydroxymethylbilane synthase (HMBS), in order to ensure that
GAPDH expression is constant in all samples. The relative
expression value for the three control genes is shown in
Supplementary Figure 2 and Supplementary Figure 3. These
results indicate that the relative expression of GAPDH to the
other control genes is fairly constant between different samples.
Furthermore, analysis of the same sample on different days
(compare TBP1, TBP2, and TBP3 in Supplementary Figure 2)
indicates that the QPCR method is reproducible.
Wes te rn bl ot
Western blotting was performed using PAGEgel precast 10%
gels and PAGEgel low molecular weight running buffer and
transfer buffer (PAGEgel, San Diego, CA, USA). Gels were
transferred to a nitrocellulose membrane and blocked with 2%
ECL advance blocking agent (GE/Amersham, Hillerød,
Denmark) for 1 hour at room temperature. Blots were incubated
with MAGE-A 6C1 monoclonal antibody (Santa Cruz Biotech,
distributed by Tebu-Bio, Roskilde, Denmark) at 1/100 dilution
overnight at 4°C, followed by goat anti-mouse HRP secondary
antibody (GE/Amersham) at 1/5000 for 1 hour at room
temperature. The blots were then washed extensively and
visualized by using ECLplus reagent (GE/Amersham) and a
Typhoon scanner to detect a chemifluorescence signal. The
www.cancerimmunity.org 11 of 11
Wei n er t et al.
MAGE-A standard curve was generated using purified
recombinant MAGE-A fusion protein (Santa Cruz Biotech).
Cell culture
DDM1.7 melanoma cells were grown in RPMI media
supplemented with glutamine and 2% human serum at 37°C
and 5% CO2. Cells were treated with 1 µM 5-aza-CdR (prepared
at 10 mM in sterile H2O, Sigma, Brøndby, Denmark) for 72 h.
Contact
Address correspondence to:
Mai-Britt Zocca
Tel.: + 45 3917 9845
Fax: + 45 3917 9900
E-mail: mbz@dandrit.com
Supplemental data
Supplementary Figure 1. Relative gene expression in normal
esophagus tissue.
Download from http://www.cancerimmunity.org/v9p9/
090909_suppl_fig1.pdf (311 KB PDF file).
Supplementary Figure 2. Housekeeping gene expression relative to
GAPDH.
Download from http://www.cancerimmunity.org/v9p9/
090909_suppl_fig2.pdf (319 KB PDF file).
Supplementary Figure 3. Housekeeping gene expression in 19 tumor
biopsies.
Download from http://www.cancerimmunity.org/v9p9/
090909_suppl_fig3.pdf (470 KB PDF file).
Supplementary Table 1. Analysis of tumor antigen gene expression
by QPCR.
Download from http://www.cancerimmunity.org/v9p9/
090909_suppl_tab1.pdf (254 KB PDF file).
Supplementary Table 2. Genes, amplified transcripts, and QPCR
primers used in this study.
Download from http://www.cancerimmunity.org/v9p9/
090909_suppl_tab2.pdf (221 KB PDF file).
Entire supplemental data set.
Download from http://www.cancerimmunity.org/v9p9/
090909_suppl_data.pdf (1.4 MB PDF file).
