Cancer
Immunity, Vol. 1, p. 4 (30 March 2001) Submitted:
16 February 2001. Accepted: 19 February 2001.
Contributed by: Lloyd J. Old
Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression
Matthew J. Scanlan1, Ivan Gout2, Claudia M. Gordon1, Barbara Williamson1, Elisabeth Stockert1, Ali O. Gure1, Dirk Jäger3, Yao-Tseng Chen1,4, Allen Mackay2, Michael J. O'Hare2, and Lloyd J. Old1
1Ludwig Institute for Cancer Research, New York Branch of Human Cancer Immunology at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 2Ludwig Institute for Cancer Research, London Branch at the University College School of Medicine, 91 Riding House Street, London W1P 8BT, England 3II.Medizinische Klinik, Hämatologie-Onkologie, Krankenhaus Nordwest, 60488 Frankfurt, Germany 4Cornell University Medical College, Department of Pathology, 1300 York Avenue, New York, NY 10021
Keywords:
Breast cancer, human, humoral immunity, tumor antigens, SEREX, mRNA, tissue distribution
Abstract
The ability of the immune system to recognize structurally altered, amplified or aberrantly expressed proteins can be used to identify molecules of etiologic relevance to cancer and to define targets for cancer immunotherapy. In the current study, ninety-four distinct antigens reactive with serum IgG from breast cancer patients were identified by immunoscreening breast cancer-derived cDNA expression libraries (SEREX). A serological profile was generated for each antigen on the basis of reactivity with allogeneic sera from normal individuals and cancer patients, and mRNA expression profiles for coding sequences were assembled based upon the tissue distribution of expressed sequence tags, Northern blots and real-time RT-PCR. Forty antigens reacted exclusively with sera from cancer patients. These included well-characterized tumor antigens, e.g. MAGE-3, MAGE-6, NY-ESO-1, Her2neu and p53, as well as newly-defined breast cancer antigens, e.g. kinesin 2, TATA element modulatory factor 1, tumor protein D52 and MAGE D, and novel gene products, e.g. NY-BR-62, NY-BR-75, NY-BR-85, and NY-BR-96. With regard to expression profiles, two of the novel gene products, NY-BR-62 and NY-BR-85, were characterized by a high level of testicular mRNA expression, and were overexpressed in 60% and 90% of breast cancers, respectively. In addition, mRNA encoding tumor protein D52 was overexpressed in 60% of breast cancer specimens, while transcripts encoding SNT-1 signal adaptor protein were downregulated in 70% of these cases. This study adds to the growing list of breast cancer antigens defined by SEREX and to the ultimate objective of identifying the complete repertoire of immunogenic gene products in human cancer (the cancer immunome).
Introduction
The structural characterization of human tumor antigens recognized by the autologous host has provided convincing evidence for immune recognition of cancer, and has led to the identification of attractive targets for vaccine-based approaches to cancer therapy (1). Innovations in immunological methods of gene discovery have led to the molecular definition of a large repertoire of tumor antigens recognized by the cellular and humoral immune systems. The discovery of MAGE antigens by the genetic method of T-lymphocyte epitope cloning (2) inaugurated current efforts to identify antigenic targets recognized by CD8+ T-lymphocytes and to develop antigen-specific cancer vaccines. A number of other human tumor antigens recognized by CD8+ T-lymphocytes have now been identified, including BAGE (3), GAGE (4), tyrosinase (5) and MelanA/Mart1 (6, 7). Progress in the identification of tumor antigens recognized by CD4+ T-lymphocytes has been hindered by the complexity of the MHC class II presentation pathway i.e., a requirement for specialized accessory molecules and processing vesicles, and the variable length of MHC class II binding peptides. In spite of these limitations, peptide epitopes recognized by CD4+ T-lymphocytes of cancer patients have been identified, including tyrosinase (8) and Melan A/Mart-1 (9), as well as mutated forms of the LDL receptor (10), CDC27, and triosephosphate isomerase (11). With regard to tumor antigens recognized by autologous human antibodies, a method referred to as SEREX (serological expression cloning) has contributed greatly to our understanding of the humoral immune response to cancer (12). To date, more than 200 SEREX-defined antigens derived from a variety of tumor types have been described in the literature (13), and an additional group of approximately 800 antigens are listed in the SEREX database (14). SEREX-defined antigens can be classified into the following categories: (a) cancer/testis (CT) antigens, e.g., MAGE-1 (12), NY-ESO-1 (15), HOM-Mel-40/SSX2 (12), CT7 (16) and SCP-1 (17); (b) mutational antigens, e.g., p53 (18) and LKB/STK11 (19); (c) overexpressed/amplified gene products, e.g., eIF-4gamma (20) and carbonic anhydrase XII (21); (d) immunogenic splice variants, e.g., NY-CO-37/PDZ-45 (18) and ING1 (22); (e) differentiation antigens, e.g., tyrosinase (12) and NY-CO-27/Galectin-4 (18); and (f) 3p antigens, encoded by putative tumor suppressor genes on chromosome 3p21.3, e.g., NY-LU-12 (23), NY-REN-9/ LUCA-15 and NY-REN-10/gene 21(19). Serum reactivity toward a number of SEREX-defined antigens, such as NY-CO-9, NY-CO-16, NY-REN-31, NY-REN-32, has been shown to be cancer-related (18, 19, 24), occurring at a variable frequency in cancer patients, but not in normal individuals. This cancer-related humoral immune response against SEREX-defined antigens suggests mutation or aberrant expression as the immunogenic stimulus, but with few exceptions, no mutations have been found and the majority of these SEREX-defined antigens are ubiquitously expressed. A recent study by Brass and colleagues (20) has provided evidence for gene amplification/overexpression as the basis for the immunogenicity of SEREX-defined antigens.
In the initial SEREX analysis of human breast cancer (22), 30 different antigens were identified, including CT antigens (HOM-Mel-40/SSX2 and NY-ESO-1), and a new member of the ING (Inhibitor of Growth) family of putative tumor suppressor proteins, ING2. In the present study, a SEREX analysis of 6 additional cases of breast cancer was carried out, and 94 breast cancer antigens were defined and characterized. In addition, the expression levels of mRNA transcripts encoding a subset of these breast cancer antigens were studied by real-time PCR in order to address the relationship between expression level and immunogenicity.
Results
Identification
of human breast cancer antigens by SEREX analysis
Serum
samples from 6 individuals with breast cancer were used to immunoscreen
tumor-derived cDNA expression libraries using the SEREX methodology.
In 4 of the 6 cases, autologous patient sera were used to screen
cDNA libraries prepared from the corresponding primary breast cancer.
In the other 2 cases, allogeneic patient sera were used to screen
a cDNA library prepared from the SK-BR-3 cell line. As shown in
Table 1, ninety-four serologically-defined breast cancer antigens
were identified. They were designated NY-BR-8 through NY-BR-101
(25). These 94 antigens (Tables 2 through 4) represent
74 known gene products and 20 novel proteins, and could be categorized
on the basis of cellular localization, subcellular targeting motifs,
known function, or sequence similarities. Thirty-nine antigens are
known or predicted nuclear proteins, and include DNA binding proteins
(e.g., BR-8, -21, -55, and -66), transcription factors (e.g., BR-11,
-52, -77, and -79), RNA binding proteins (e.g., BR-17, -18, -74
and -91), and RNA/DNA helicases (BR-65, -78 and -82). Twenty-four
antigens are known or predicted cytoplasmic proteins, and include
metabolic enzymes (e.g., BR-28, -30, -39 and -58), ribosomal proteins
(e.g., BR-31, -32, -64 and -92), cytoskeletal proteins (BR-10, -88
and -101), sorting proteins (e.g., BR-42, -44, -48, and -80), adaptor
proteins (BR-34 and BR-41) and proteins involved in the ubiquitin
pathway (BR-29 and BR-37). Six antigens localize to the plasma membrane
(BR-27, -43, -56, -57, -82 and -90) and 2 are extracellular gene
products (BR-35 and BR-36). Twenty-three antigens had no defining
structural characteristics. The nucleotide sequences of all novel
clones have been deposited in the GenBank database (sequential accession
numbers AF308284 - AF308300). The cDNA sequences coding for the
94 antigens identified in this study were also compared to sequences
deposited in the SEREX database (14).
Examination of this database revealed that 25 of the 94 breast cancer
antigens defined in this study (26%) were also identified through
SEREX analysis of breast cancer and other tumor types (Tables 2,
3 and 5).
SEREX-defined
breast cancer antigens: Antigens reactive with sera from
breast cancer patients and patients with other forms of
cancer
Reactivity
patterns of sera from normal individuals and cancer patients with
SEREX-defined breast cancer antigens
To determine whether immune recognition of the isolated antigens
was cancer-related, allogeneic sera samples obtained from 25 normal
blood donors and 24 additional patients with breast cancer were
tested for reactivity against the panel of antigens using the
plaque assay. Fifty-four of the 94 antigens (57%) had a serological
profile that was not restricted to cancer patients, as evidenced
by their reactivity with normal sera (Tables 1 and 2) or their
known association with autoimmune disease (Tables 1 and 3). The
remaining 40 antigens had a cancer-related serological profile,
reacting only with sera from cancer patients (Tables 1, 4 and
5).
The
40 antigens having a cancer-related serological profile were further
tested for reactivity with serum panels from colon, lung, ovarian
and esophageal cancer patients using the spot assay shown in Figure
1, and the following seroreactivity patterns were defined: (a) antigens
identified by serum from only a single breast cancer patient; (b)
antigens that reacted exclusively with sera from 2 or more breast
cancer patients; and (c) antigens that reacted with sera from patients
having other forms of cancer in addition to breast cancer patients.
Of the 40 antigens having a cancer-related serological profile,
24 antigens reacted with sera from only one breast cancer patient,
either the autologous patient or a single allogeneic patient, and
are listed in Table 4. Sixteen of the 40 antigens having a cancer-related
serological profile reacted with sera from more than one cancer
patient (breast cancer patients and/or patients with other tumor
types), and are listed in Table 5. Four of these antigens [gC1Q/BR-56,
Her2neu/BR-57, BR-62, and tumor protein D52 (TPD52)/BR-63] reacted
with sera from more than one breast cancer patient, but not with
sera from patients with other forms of cancer. The remaining antigens
listed in Table 5 react with sera from both breast cancer patients
and individuals with other forms of cancer. In terms of known tumor
antigens, LAGE-1/BR-53, a cancer-testis antigen highly homologous
to NY-ESO-1, was isolated in the current study. In accordance with
results of previous serologic surveys (24),
LAGE-1/NY-ESO-1 had the highest frequency of cancer-related seroreactivity;
antibody responses were detected in patients with breast (4/20),
colon (1/19), lung (2/15), ovarian (1/15) and esophageal cancer
(2/15). Similarly, a cancer-related serological response was also
detected against p53/BR-94 (1/25 breast cancer, 3/19 colon cancer,
1/15 ovarian cancer and 2/15 esophageal cancer patients), and Her2neu/BR-57
(2/25 breast cancers patients), confirming previous surveys of seroreactivity
against these antigens (18,
26,
27).
In addition, a new set of immunogenic breast cancer antigens were
defined in this study, including known proteins such as kinesin
2/BR-45, gC1Q binding protein/BR-56, TPD52/BR-63 and MAGE D/BR-98,
as well as novel gene products such as NY-BR-62, NY-BR-75, and NY-BR-96.
Nitrocellulose
spot assay of duplicate bacteriophage clones expressing
44 different serologically-defined breast cancer antigens.
Following incubation in a 1:200 dilution of serum from
a colon cancer patient, the membrane was exposed to alkaline
phosphatase-conjugated, Fc fragment-specific, goat anti-human
IgG. A positive color reaction with 4-nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl-phosphate indicates
seroreactivity with nucleosome assembly protein-1/NY-BR-55,
NY-BR-87 and p53/BR-94, as well with the positive control
antigen PINCH (PCH)/NY-CO-22.
Expression
patterns of mRNA encoding serologically-defined breast cancer
antigens in normal tissues
A preliminary in silico mRNA expression profile of all
gene products identified in this study was based on the tissue
distribution of expressed sequence tags (ESTs) in the human EST
database. Products with no EST matches or those having EST matches
limited to tumor tissue, fetal tissue or a single normal tissue
were further examined by Northern blotting and RT-PCR. Gene products
with restricted EST profiles include the three well-characterized
cancer-testis antigens, MAGE A3/BR-76, MAGE A6/ BR-72 and LAGE-1/BR-53,
which are expressed exclusively in normal testis and a range of
different tumor types (13), and 4 putative tissue
restricted antigens, including a known gene product, SNT-1/BR-41,
and 3 novel proteins, BR-49, -62 and -85. All seven of these antigens
showed a cancer-related serological profile.
As shown in Figure 2A, the mRNA expression profiles of BR-41,
-49, -62 and -85 were examined in Northern blots of non-normalized
mRNA preparations derived from 16 different human tissues. Expression
of SNT-1/BR-41, BR-62 and BR-85 was restricted to testis, appearing
as a 3.2 kb mRNA transcript, a 4.0 kb mRNA transcript, and 2.4
kb and 3.2 kb mRNA transcripts, respectively. Expression of BR-49
was widespread, appearing as a 4.2 kb hybridization signal in
10 of 16 normal tissues (absent in spleen, ovary, PBL, heart,
brain and skeletal muscle). The mRNA expression patterns of BR-41,
-49, -62 and -85 were also analyzed by conventional RT-PCR, and
transcripts for these 4 gene products were found in all normal
tissues tested (lung, testis, small intestine, breast, liver,
and placenta).
mRNA
expression pattern of four antigens associated with a
cancer related serological response. (A) Northern
blot analysis of NY-BR-41, -49, -62 and -85 mRNA. Testis
restricted expression was observed for NY-BR-41, -62 and
-85, while NY-BR-49 was expressed in 10/16 normal tissues.
(B) Real-time RT-PCR analysis of the level of NY-BR-41
mRNA expression (fg of homologous cDNA ± SEM).
Results show low level ubiquitous expression. Highest
levels of NY-BR-41 mRNA were detected in testis (43.8
± 0.83 fg) and peripheral blood leukocytes (35.4
± 2.65 fg), while lower levels (less than 18.0
fg) were detected in the remaining 12 normal tissues.
(C) Real-time RT-PCR analysis of mRNA encoding NY-BR-62
showing low level ubiquitous expression (fg of homologous
cDNA ± SEM). Highest levels of NY-BR-62 mRNA expression
were detected in testis (270.7 ± 7.13 fg ), thymus
(88.8 ± 7.89 fg), and colon (20.2 ± 1.40
fg), while low levels (less 6.0 fg) were observed in 11
other normal tissues. (D) Real-time RT-PCR analysis of
mRNA encoding NY-BR-85 showing low level ubiquitous expression
(fg of homologous cDNA ± SEM). Expression of NY-BR-85
mRNA was detected in thymus (54.5 ± 6.70 fg ),
testis (38.8 ± 2.61 fg), colon (15.7 ± 0.38
fg), spleen (15.0 ± 1.04 fg) and peripheral blood
leukocytes (12.5 ± 0.72 fg), while lower levels
(less 10 fg) were detected in each of the remaining 9
normal tissues.
The detection of BR-41, -62 and -85 mRNA in normal tissues by
RT-PCR but not by Northern blotting indicates low level ubiquitous
expression. To examine this further, real-time RT-PCR was used
to quantify mRNA expression in a panel of normalized cDNA preparations
from 15 different human tissues. PCR amplification of cDNA coding
for BR-41, -62 and -85 yielded distinct values for cycle threshold
(Ct), which were compared to the Ct values obtained from a set
of homologous cDNA standards of known concentration. As shown
in Figure 2B, the highest levels of BR-41 mRNA expression in the
normalized cDNA panel were detected in testis (equivalent to 43.8
fg of cDNA) and peripheral blood leukocytes (equivalent to 35.4
fg of cDNA), and lower levels (less than 18.0 fg of cDNA) in each
of the remaining 12 normal tissues. Since cDNA from normal breast
tissue was not included in this commercially obtained cDNA panel,
the concentration of BR-41 mRNA in normal mammary gland was calculated
relative to normal testis, and determined to be approximated 170
times higher in normal breast tissue compared to testis, and was
equivalent 7300 fg of cDNA. The highest levels of BR-62 mRNA expression
were detected in normal testis (271 fg of cDNA), thymus (89 fg
of cDNA), and colon (20 fg of cDNA), with lower levels (less 6
fg of cDNA) detected in each of the remaining 11 normal tissues.
The concentration of BR-62 mRNA in normal breast was equivalent
to 0.8 fg of cDNA. The highest levels of BR-85 mRNA expression
were detected in normal thymus (54 fg of cDNA) and testis (39
fg of cDNA), while lower levels (less than 16 fg of cDNA) were
detected in each of the remaining 12 normal tissues. The concentration
of BR-85 mRNA in normal breast was equivalent to 8.0 fg of cDNA.
Expression levels of mRNA encoding serologically-defined breast
cancer antigens in normal and malignant breast tissue
The mRNA expression levels of ten antigens associated with a cancer-related
serological response (Table 5) and/or showing differential mRNA
expression (Figure 2) were measured in 10 breast cancer specimens
and normal breast tissue by real-time RT-PCR. The relative level
of mRNA expression in breast cancer compared to normal breast
tissue was calculated on the basis of differences in normalized
Ct values between the 2 tissues. Overexpression was defined as
an mRNA expression level that is 3 times higher in tumor compared
to normal breast tissue. As shown in Table 6, mRNA transcripts
encoding 6/10 antigens, BR-56, -57, -62, -63, -85 and -98, were
overexpressed in at least 1 breast cancer specimen. Four of these
10 antigens were frequently overexpressed in breast cancer, including
Her2neu/BR-57 (3/10 cases), BR-62 (6/10 cases), TPD52/BR-63 (5/10
cases), and BR-85 (9/10 cases). Exceptionally high levels of mRNA
expression in breast cancer were associated with Her2neu/BR-57
(107 times that of normal breast in patient # 7), BR-62 (53 times
that of normal breast in patient # 8) and TPD52/BR-63 (132 times
that of normal breast in patient # 8). In contrast, expression
of SNT-1/BR-41 mRNA was consistently lower in breast cancer relative
to normal breast tissue, with apparent downregulated expression
(defined as an expression level of less than 20% of normal breast)
occurring in 7/10 cases. The remaining products, kinesin 2/BR-45,
BR-75 and BR-96, had similar mRNA expression levels in tumor and
normal breast tissue.
Quantitative
analysis of mRNA encoding SEREX-defined breast cancer
antigens in normal and malignant breast tissue: Relation
between mRNA level and immunogenicity
The relationship between upregulation of gene expression and serological
reactivity was examined in 8 breast cancer patients whose tumors
and sera were available for typing (informative cases, patients
# 1, 3, 5, 6, 7, 8, 9, 10). Serum antibodies against BR-56, -57,
-62, -63, -75 and -85 were detected in breast cancer patient #10.
In this case, mRNA transcripts encoding 4/6 of these antigens
were overexpressed in the autologous tumor (BR-56, -62, -63 and
-85). None of the other cases showed seroreactivity against the
amplified/overexpressed SEREX-defined antigens.
Discussion
Knowledge regarding the structure, expression pattern, and immunogenicity
of serologically-defined cancer antigens is critical in assessing
their relevance to cancer and their therapeutic and diagnostic
potential. Given the large number of tumor antigens defined by
SEREX analysis, a strategy needed to be developed that could identify
antigens of the greatest interest in the context of cancer. Although
mutational changes or cancer-restricted expression would be properties
of obvious importance, these turn out to be characteristics that
are rarely associated with SEREX defined antigens. For this reason,
we have chosen to focus our attention on the frequency of antibody
responses to the antigens in normal individuals and cancer patients,
selecting those antigens with cancer-related serological profiles
for detailed study. In terms of allogeneic serum reactivity, 54
of the 94 antigens identified in the current study had a serological
profile that was not restricted to cancer patients. Although this
provides presumptive evidence that these antigens are unrelated
to cancer, they should not be dismissed without further study
since some of these antigens have functional properties associated
with tumor growth, such as angiogenesis [e.g. vascular endothelial
cell growth factor/BR-35 (28)],
anti-apoptosis [e.g. defender against death protein-1/BR-43 (29)],
and regulation of cell proliferation [e.g. Zuotin related factor
1/BR-13 (30)].
The remaining 40 antigens were characterized by a cancer-related
serological profile, reacting only with sera from cancer patients
and not with sera from normal individuals. These include known
tumor antigens such as LAGE-1/BR-53 (31),
Her2neu/BR-57 (26),
MAGE-A6/BR-72 (32),
MAGE-A3/BR-76 (33)
and p53/BR-94 (27),
as well as several transcriptional regulators such as RING 3/BR-11
(34),
modulator recognition factor/BR-26 (35),
SNT-1 adaptor protein/BR-41 (36),
Snf2-related CBP activator protein/BR-52 (37),
SMRT corepressor/BR-77 (38)
and TATA modulatory factor 1/BR-79 (39).
With regard to novel antigens identified in this study, 11 were
associated with a cancer-related serological profile, and their
significance in the context of cancer is currently being investigated.
In conformity with previous results, a small subset of antigens
that show a cancer-related serological profile are characterized
by differential gene expression; these include the CT antigens,
LAGE-1/BR-53, MAGE-6/BR-72 and MAGE-3/BR-76, and two novel antigens,
BR-62 and BR-85. The BR-62 cDNA encodes a 312 amino acid protein
which contains a bZIP transcription factor domain and is 48% identical
to Xklp2, a kinesin-like protein required for mitotic centrosome
separation in Xenopus laevis (40).
The BR-85 cDNA encodes a 278 amino acid protein containing two
bipartite nuclear localization signals and lacking any similarity
with proteins in the existing databases. Although Northern blots
indicated that BR-62 and BR-85 expression is restricted to testis,
quantitative RT-PCR analysis of these antigens showed ubiquitous,
low level expression in several normal tissues. In tumor tissue,
BR-62 mRNA was found to be overexpressed in 60% of breast cancer
specimens at levels that were approximately 4-50 times higher
than normal breast tissue, while BR-85 mRNA was overexpressed
in 90% of breast cancer specimens at levels that were approximately
3-11 times higher than normal breast tissue. Transcripts encoding
4 other antigens with cancer related-serological profiles, Her2neu/
BR-57, TPD52/BR-63, gC1Q/BR-56 and MAGE D/BR-98, were overexpressed
in 30%, 50%, 10% and 10% of the breast cancer specimens, respectively.
As bulk tumor and normal tissue were used in these studies, the
precise cell type responsible for overexpression is not known
and requires detailed investigation. TPD52/BR-63, which has been
reported to be overexpressed in breast (41)
and lung cancer (42),
was found to be immunogenic in 2/25 breast cancer patients. This
protein is thought to be involved in calcium-mediated signal transduction
and the regulation of cell proliferation (41),
suggesting that its upregulation is of possible etiologic significance.
BR-56/gC1q binding protein, which was found to be immunogenic
in 2/25 breast cancer patients, is a multifunctional protein that
interacts with the globular domain of complement component C1q
at the cell surface (43),
and is a component of the pre-mRNA splicing factor, SF2 (44).
Unpublished reports (GenBank entries) concerning MAGE D/BR-98
indicate that it is overexpressed in breast and hepatocellular
cancer. MAGE D/BR-98 was associated with a serological response
in patients with breast (3/25) and lung cancer (1/15). Patients
with serological reactivity toward MAGE D lacked detectable antibodies
against MAGE A3 and MAGE A6, indicating that isolation of MAGE
D/BR-98 by SEREX is not a result of cross-reacting antibodies
to other MAGE antigens. Although MAGE D/BR-98 appears to be a
member of the MAGE gene family, it is ubiquitously expressed (contrary
to the cancer/testis expression pattern of other MAGE genes) and
does not contain any of the CTL-defined epitopes previously described
for members of this antigen family (45).
In contrast to SEREX-defined gene products showing overexpression
in breast cancer, expression of SNT-1/BR-41 appears to be markedly
downregulated in 70% of breast cancer specimens relative to normal
breast tissue. SNT-1 /BR-41 is a membrane-associated adaptor protein
that undergoes rapid tyrosine phosphorylation following stimulation
with growth factors and interacts with the Ras guanine nucleotide
exchange factor, SOS. Given its potential role in signal transduction
pathways (36),
downregulated expression of SNT-1/BR-41 may have significance
in relation to cancer. In this regard, how does a protein having
reduced levels in cancer induce an immune response? One possibility
is that the immune system has the capacity to respond to downregulated
as well as upregulated (overexpressed/amplified) epitopes. The
SEREX-defined "3p" antigens, NY-LU-12 (23),
NY-REN-9/ LUCA-15 (19),
NY-REN-10/gene 21(19)
and NY-BR-79/TATA modulatory factor-1, represent other examples
of an immune response to gene products that are deleted or downregulated
in cancer. These antigens map to the putative tumor suppressor
gene locus on 3p21, a region often deleted (downregulated) in
small cell lung cancer (46),
breast cancer (47)
and renal cancer (48).
Brass and colleagues (20)
have reported a striking example of several overexpressed/amplified
gene products associated with antibody responses in patients with
lung cancer and suggested that this upregulated expression was
the basis for the immunogenicity of these antigens. One patient
in our breast cancer series (patient #10) also had a strong association
between overexpression and immune response, with an antibody response
to 4 of 6 overexpressed gene products in the autologous tumor.
However, other patients failed to mount an immune response to
overexpressed products in their tumors, indicating that overexpression
is not an invariable prelude to an antibody response.
In addition to quantitative changes in gene expression (overexpression/amplification,
underexpression/loss) being involved in the immunogenicity of
SEREX-defined antigens, examples of qualitative changes eliciting
an antibody response, although uncommon, are also known. These
include aberrant expression of tissue restricted gene products,
e.g., CT antigens, and structural changes, e.g., mutation, abnormal
splice variants, and altered reading frames. With regard to the
latter, the novel antigen, NY-BR-87, has two possible reading
frames of 202 amino acids or 205 amino acids. Given the immunogenicity
of BR-87, it would be important to determine whether expression
of an alternative BR-87 reading frame in cancer cells can elicit
an immune response. Immunogenic alternative reading frames have
been described for tumor antigens such as NY-ESO-1 (49)
and RU2AS (50).
Serological analysis with purified recombinant proteins representing
both reading frames of BR-87, as well as the development of monoclonal
antibodies to the two putative proteins and their subsequent use
in immunohistochemistry will clarify this possibility. One of
the more perplexing aspects of SEREX analysis is how rarely mutated
gene products are isolated. Products of mutational events may
in fact be the immunogenic stimulus for initiating a humoral immune
response, but the resulting antibody reacts predominately with
non-mutated protein sequences. This appears to be the case for
the immunogenicity of p53 in patients with cancer (51),
although it is possible that overexpression plays a role in antibody
responses to p53, since mutations in p53 increase its half life
and leads to p53 accumulation in tumor cells. A consequence of
the possible predilection of the antibody response to focus on
non-mutated sequences in mutated gene products is the likelihood
that the non-mutated gene counterpart will be isolated by SEREX,
derived from a non-mutated allele in the cancer cell, or from
normal admixed cells in the tumor mass. To exclude this latter
possibility, isolated tumor cell populations or tumor cell lines
should be employed as SEREX targets. Clearly, much more attention
needs to be given to the possibility that mutation is a primary
reason for the immunogenicity of SEREX-defined antigens, and approaches
such as single-strand conformation polymorphism, dideoxy fingerprinting,
or direct sequence analysis of multiple clones (both cDNA and
genomic) need to be included in the analysis of these gene products.
Antigens that map to loci implicated in human cancer are prime
candidates for further mutational analysis, and include BR-79/TATA
modulatory factor-1, a coactivator of the androgen receptor (39),
which maps to 3p21.3, a site of minimal deletion in breast (47),
renal (48)
and non-small cell lung cancer (46),
as well as BR-81, which maps to chromosome 6q23.3 (D6S311-D6S440),
a site of minimal deletion in breast (52),
ovarian (53)
and cervical cancers (54).
We thank Ms. Latasha Hasty of the Ludwig Institute for Cancer
Research, New York Branch, for her excellent technical assistance.
The SEREX program of the Ludwig Institute for Cancer Research
provided financial support for the current study.
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Materials
and methods
Construction
of cDNA libraries
Tumor samples from breast cancer patients 184 (invasive ductal
carcinoma), 297 (invasive ductal carcinoma), 257 (invasive ductal
carcinoma), and 263 (invasive pleomorphic lobular carcinoma) were
obtained as surgical specimens and used in the construction of
cDNA libraries. A cDNA library was also prepared from the SK-BR-3
breast cancer cell line (ATCC HTB-30). Total RNA was prepared
by the guanidinium thiocyanate method and purified to poly(A)+
RNA using the Dynabeads mRNA purification Kit (Dynal, Lake Success,
NY). Manufacturer's protocols were used for cDNA synthesis and
ligation into appropriate vectors. Two bacteriophage expression
vectors were utilized; the lambda ZAP vector (Stratagene, La Jolla,
CA) was used for cDNA derived from the tumor of patient 184 and
the SK-BR-3 cell line, and the lambda TRIPLX vector (Clontech
laboratories Inc., Palo Alto, CA) was used for cDNA derived from
the tumors of patients 297, 257 and 263. Following in vitro
packaging, libraries containing 1-2 x 106 primary recombinants
were obtained. cDNA libraries were not amplified prior to immunoscreening.
Immunoscreening
Sera were obtained from individuals undergoing diagnostic or therapeutic
procedures. To remove antibodies reactive with vector-related
antigens, sera (1:10 dilution) were absorbed by passage through
columns containing Sepharose 4B coupled to lysates of E. coli
Y1090 and bacteriophage-infected E. coli BNN97 (5 Prime
3 Prime, Inc., Boulder, Co.), followed by a 15 hour incubation
with nitrocellulose filters precoated with proteins derived from
E. coli and E.coli/phage lysates (mock experimental
membranes). Library screenings were performed as previously described
(18,
19)
using preadsorbed patient sera at a dilution of 1:200. A total
of 5-6 x 105 recombinants were screened per cDNA library.
Serum reactive phage clones were converted to plasmid forms by
in vivo excision. Plasmid DNA was sequenced at the Cornell
University DNA Service (Ithaca, NY) using an ABI Prism automated
DNA sequencer (Perkin Elmer, Foster City, CA).
Two assays were employed to determine allogeneic serum reactivity.
In the case of allogeneic sera derived from breast cancer patients
and normal individuals, a previously described plaque assay was
used (18,
19).
Briefly, 1500 plaque-forming units (pfu) of monoclonal phage encoding
individual serologically-defined breast cancer antigens were mixed
with an equal number of negative control phage (phage without
cDNA inserts) and used to infect exponentially growing E. coli
XL-1 Blue MRF' (Stratagene). Following a 15 hour amplification
phase, plaque-derived proteins were transferred to 47 mm nitrocellulose
membrane discs and tested for reactivity with individual serum
samples (1:200 dilution) as previously described (18,
19).
Subsequent screening of serum samples from patients with other
forms of cancer (lung, colon, ovarian, esophageal) was carried
out using a modification of the plaque assay, termed a spot assay.
In this method, 80 x 120 mm nitrocellulose membranes were precoated
with a film of NZY/0.7% Agarose/2.5 mM IPTG and placed on a reservoir
layer of NZY/0.7% Agarose in a 86 x 128 mm Omni Tray (Nalge Nunc
International Corp., Naperville, IL). Approximately 1.0 x 105
pfu of monoclonal phage encoding individual serologically-defined
breast cancer antigens, in a volume of 20 µl, were mixed
with 20 µl of exponentially growing E. coli XL-1
Blue MRF and spotted (0.7µl aliquots) on the precoated nitrocellulose
membranes. Membranes were incubated for 15 hours at 37°C.
A total of 46 different serologically-defined breast cancer antigens
were spotted in duplicate per nitrocellulose membrane. The agarose
film was then removed from the membrane and the filters were processed
for reactivity with individual serum samples (1:200 dilution)
as previously described (18,
19).
Serum reactivity detected by the spot assay was verified in the
plaque assay. Both assays appear to have comparable specificity
and sensitivity.
Northern blot analysis and standard RT-PCR
Northern blots containing normal tissue poly(A)+ RNA
(2 µg/lane) were obtained from Clontech, laboratories Inc.
(Palo Alto, CA). Random-primed 32P-labeled probes consisting
of 300-600 bp PCR products from coding sequences of selected seroreactive
cDNA clones were hybridized for 1.5 hours in Expresshyb (Clontech)
at 68°C, and washed at high stringency (2 times, 30 min each,
with 0.1X SSC/0.1% SDS at 65°C). The resultant Northern blots
were developed with Biomax MS autoradiography films (Eastman Kodak
Co., Rochester, NY).
The mRNA expression pattern of a selected set of serologically-defined
breast cancer antigens was also determined by standard RT-PCR,
using a panel of normal tissue RNA from lung, testis, small intestine,
breast, liver, and placenta (Clontech). cDNA preparations used
as templates in the RT-PCR reactions were synthesized using MuLV
reverse transcriptase as described previously (19).
As a control for genomic DNA contamination, all cDNA synthesis
reactions were set up in duplicate with additional samples lacking
reverse transcriptase. The cDNA was then amplified by PCR (30
cycles), using gene-specific primers (Gibco BRL, Grand Island,
NY) and AmpliTaq Gold DNA polymerase (PE Biosystems), as described
(19).
Real-time quantitative RT-PCR
Total RNA from 10 different breast cancer specimens was prepared
by the guanidinium thiocyanate method. Total RNA from normal breast
consisted of a pool of RNA from 2 healthy individuals (Clontech).
RNA (1 µg) was reverse transcribed into cDNA using the TaqMan
EZ RT-PCR kit (PE Biosystems, Foster City CA). As a control for
genomic DNA contamination, all cDNA synthesis reactions were set
up in duplicate with additional samples lacking reverse transcriptase.
Reagents were purchased from PE Biosystems, except where noted.
Multiplex PCR reactions were prepared using 2.5 µl of cDNA
diluted in TaqMan Universal PCR Master Mix supplemented with Vic-labeled
(PE Biosystems proprietary dye) human beta-glucuronidase endogenous
control probe/primer mix, 200 nM 6-carboxy-fluorescein (Fam)-labeled
gene-specific TaqMan probe, and a predetermined, optimum concentration
of the corresponding gene-specific forward and reverse primers
(300-900 nM). Triplicate PCR reactions were prepared for each
cDNA sample. PCR consisted of 40 cycles of 95°C denaturation
(15 seconds) and 60°C annealing/extension (60 seconds). Thermal
cycling and fluorescent monitoring were performed using an ABI
7700 sequence analyzer (PE Biosystems). The point at which the
PCR product is first detected above a fixed threshold, termed
cycle threshold (Ct), was determined, and normalized against the
Ct value of the endogenous control product (DELTACt = Ct Fam -
Ct Vic). The quantity of gene-specific transcripts present in
the breast cancer cDNA samples relative to normal breast tissue
was calculated by comparing the normalized cycle thresholds in
tumor with non-malignant breast tissue (DELTADELTACt = DELTACt
of tumor - DELTACt of normal breast), and determining the relative
concentration (Relative Concentration = 2-DELTADELTACt).
In addition, a selected set of Fam-labeled gene-specific TaqMan
probe/primer combinations were used to amplify a panel of 14 normal
tissue cDNA preparations which had been normalized against 6 housekeeping
genes and purported to be virtually free of genomic DNA (Clontech).
The relative abundance of gene-specific transcripts in normal
tissues was determined by comparison with a standard curve generated
from the Ct values of known concentrations of plasmid DNA containing
the relevant gene. Since cDNA derived from normal breast tissue
was not included in the commercially obtained panel, a cDNA preparation
derived from normal breast tissue (see above) was normalized according
to the Ct values for beta-glucuronidase, and the concentration
of gene-specific transcripts in normal breast tissue was also
calculated relative to its expression in testis using the formula
described above.
Contact
Address
correspondence to:
Matthew J. Scanlan
Ludwig Institute for Cancer Research
New York Branch of Human Cancer Immunology at Memorial Sloan-Kettering
Cancer Center
1275 York Avenue
New York, NY 10021
USA
Fax: + 1 212 639 84 81
E-mail:
