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Autoantibody Cancer Biomarker: Extracellular Protein Kinase A
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1
1
2
4
Maria V. Nesterova, Natalie Johnson, Christopher Cheadle, Susan E. Bates, Sridhar Mani,
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5
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1
Constantine A. Stratakis, Islam Kahn, Rishab K. Gupta, and Yoon S. Cho-Chung
1
Cellular Biochemistry Section, Basic Research Laboratory, 2Medical Oncology Branch, Center for Cancer Research, National Cancer
Institute; 3National Institute of Child Health and Human Development, NIH, Bethesda, Maryland; 4Montefiore Medical Center, Albert
Einstein College of Medicine, Bronx, New York; 5Wake Forest University School of Medicine, Winston-Salem, North Carolina;
and 6John Wayne Cancer Institute, Santa Monica, California
Abstract
In cancer cells, cyclic AMP–dependent protein kinase (PKA) is
secreted into the conditioned medium. This PKA, designated as
extracellular protein kinase A (ECPKA), is markedly upregulated in the sera of patients with cancer. The currently
available tumor markers are based on the antigen determination method and lack specificity and sensitivity. Here, we
present an ECPKA autoantibody detection method for a
universal biomarker that detects cancer of various cell types.
We tested sera from 295 patients with cancers of various cell
types, 155 normal controls, and 55 patients without cancer.
The specificity and sensitivity of this autoantibody enzyme
immunoassay method were compared with the conventional
antigen determination method by receiver-operating characteristic plots. In the sera, the presence of autoantibody directed
against ECPKA was highly correlated with cancer. High antiECPKA autoantibody titers ( frequency, 90%; mean titer, 3.0)
were found in the sera of patients with various cancers,
whereas low or negative titers ( frequency, 12%; mean titer, 1.0)
were found in the control group. The receiver-operating
characteristic plot showed that autoantibody enzyme immunoassay exhibited 90% sensitivity and 88% specificity, whereas
the enzymatic assay exhibited 83% sensitivity and 80%
specificity. These results show that the autoantibody method
distinguished between patients with cancer and controls better
than the antigen method could. Our results show that
autoantibody ECPKA is a universal serum biomarker for
cancers of various cell types. (Cancer Res 2006; 66(18): 8971-4)
Introduction
Early detection and diagnosis are of utmost importance in
cancer management. Serum biomarker measurement in body fluid
immunoassays has been the most widely used approach, generally
of such established tumor-associated markers as CEA, AFP, hCG,
PSA, and CA125. The lack of tumor specificity of these markers,
however, precludes their general use in cancer screening and
diagnosis (1–5). The limitations of the presently available serum
tumor markers, based on the antigen determination method,
indicate the need for other means of screening (1–5).
In normal mammalian cells, cyclic AMP–dependent protein
kinase (PKA) is present strictly intracellularly (6). Intriguingly,
however, cancer cells of various types excrete PKA into the
conditioned medium (7, 8). This PKA, designated as extracellular
Requests for reprints: Yoon S. Cho-Chung, National Cancer Institute, Building 10,
Room 5B05, 9000 Rockville Pike, Bethesda, MD 20892-1750. Phone: 301-496-4020; Fax:
301-480-8587; E-mail: [email protected].
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-1049
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PKA (ECPKA) was found to be markedly up-regulated in the serum
of patients with cancer (7, 8), and surgical removal of tumors led to
a decrease in ECPKA levels in patients (9). Two types of PKA exist,
designated type I (PKA-I) and type II (PKA-II; ref. 6); they are
distinguished by different regulatory (R) subunits (RI and RII), and
they contain a common catalytic (C) subunit (10). Importantly, the
ratios of PKA-I to PKA-II change dramatically during cell development, differentiation, and transformation (11). In cancer cells,
ECPKA expression was modulated by changing the ratios of the
intracellular PKA-I to PKA-II (7), and down-regulation of ECPKA
was shown by a decrease in PKA-I and by a mutant Ca lacking the
NH2-terminal myristyl group (7). These results indicate that
ECPKA might be a cancer antigen. There is increasing evidence
that patients with cancer produce autoantibodies against antigens
in their tumors (12–15), suggesting that such autoantibodies could
have diagnostic/prognostic value. We speculated that ECPKA
excretion might elicit the induction of serum autoantibodies and
that the presence of such autoantibodies could serve for cancer
detection. For this, we developed a novel enzyme immunoassay
(EIA) that measures the anti-IgG autoantibody for ECPKA.
Materials and Methods
We tested 295 serum samples from patients with cancer. The cancers
included various cell types: breast (n = 24), cervical (n = 13), colon (n = 40),
lung (n = 6), ovarian (n = 28), prostate (n = 35), pancreatic (n = 6), renal
cell (n = 60), and rectal (n = 14) carcinomas, melanomas (n = 50), and other
carcinomas (n = 19), including bladder, esophageal, gastric, hepatocellular,
and small bowel; and sarcoma, thymoma, liposarcoma, and leiomyosarcoma. As controls, we used normal sera from a blood bank (n = 155) and sera
from patients with diseases other than cancer (n = 55; systemic lupus
erythematosus, 20; Carney complex, 25; other diseases, 10).
The sera used for this autoantibody EIA assay were from existing
individual Institutional Review Board–approved, consented samples, and
were therefore not drawn from patients for the specific studies presented
here. The EIA autoantibody assay was not done to distinguish the stage and
site of malignancy but was done in the sera of patients with a wide range of
active malignancies. For comparison, the same samples were also analyzed
with the conventional PKA assay (antigen determination).
The serum samples were aliquoted (5 AL volume) and kept frozen at
80jC until use. The serum samples were thawed only once before use,
and these diluted serum samples were never used twice. The autoantibody
levels were stable over 6 months.
EIA method. Anti-ECPKA IgG autoantibodies were measured by solid
phase EIA. The plates were coated with 100 AL of diluted antigen (2 Ag/mL
concentration in PBS) of the purified recombinant human PKA Ca subunit
(7), and were incubated overnight at room temperature. The plates were then
washed once with washing buffer [20 mmol/L Hepes, 0.9% NaCl,
30 mmol/L sucrose, 0.1% bovine serum albumin, BSA (pH 7.0)], blocked for
2 hours at room temperature with 100 AL of Blockase (Serotec),7 and washed
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http://www.serotec.com.
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twice with sodium citrate washing solution [50 mmol/L sodium citrate,
0.15 mol/L NaCl, and 0.1% Tween 20 (pH 5.0-5.2)]. We then added 100 AL of
25,000-fold diluted serum samples [dilution buffer: PBS (pH 7.4), 0.25% BSA
( fatty acid–free fraction V), and 0.05% Tween 20] and incubated the plates for
1 hour at 37jC. After three washes with sodium citrate solution, we added 100
AL of 20,000-fold diluted anti-human IgG-horseradish peroxidase antibodyenzyme conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA),
in PBS, 0.9% NaCl, and 1% BSA, incubated the plates for 1 hour at room
temperature, then washed them five times in sodium citrate solution and
added 100 AL of TMB substrate. The reaction was stopped with 100 AL of 0.45
mol/L H2SO4 reagent and the absorbance was read at 450 nm and recorded
on an ELISA reader (microplate reader benchmark; Bio-Rad, Hercules, CA).
Purification of PKA CA. The recombinant human PKA Ca (1.1 kb) from
the OT1529-Ca plasmid (7) was infused with pQE31 DNA leading to the
production of pQE31-Ca (S.H. Hong, Seoul National University, Seoul,
Korea). pQE31-Ca plasmid was expressed in Escherichia coli, and native
PKA Ca protein was purified (Paragon, Baltimore, MD).
Western blotting analysis of anti-ECPKA autoantibody. The purified,
recombinant human Ca protein (1 Ag/lane) was subjected to 10%
SDS-PAGE, and electroblotted onto nitrocellulose membrane. The membrane were blocked, washed, and the blot strips were incubated with
patients’ or normal sera diluted in Tween 20 PBS solution, the strips were
washed, then incubated with anti-human IgG-horseradish peroxidase
conjugate, and immunodetection was done by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).
ECPKA enzymatic assay. The serum ECPKA activity was measured as
previously described (7). One unit of PKA activity was defined as the
amount of enzyme that transferred 1 pmol of 32P from (g-32P)ATP to
recovered protein in 1 minute at 37jC in the standard assay system.
Statistical analysis. Means, SD, and confidence intervals were used
where appropriate. Data on the reproducibility of the EIA test was analyzed
by analytic coefficients of variation at relevant concentrations and
for appropriate time intervals; we also measured intraobserver and
interobserver variability. P < 0.05 were considered significant throughout.
Receiver-operating characteristic (ROC) curves (16) were used to calculate
cutoff values for optimal sensitivity and specificity.
The ECPKA autoantibody levels were also evaluated in the sera
of patients with diseases other than cancer. Patients with systemic
lupus erythematosus, an autoimmune disease that accompanies
marked decrease in intracellular PKA (17); Carney complex, an
adrenal gland disorder causatively related to mutational loss of the
PKA RIa subunit (18); and patients with diseases other than
cancer, including pancreatitis, angina, and hypertension, exhibited
average ECPKA autoantibody titers of 1.3, 0.8 (Fig. 1), and 1.2
(data not shown), respectively, levels close to that of normal
controls (1.0; Fig. 1). These results show that the autoantibody
ECPKA is elevated in patients with a wide range of active
malignancies of various cell types (see Materials and Methods).
Comparison between ECPKA autoantibody EIA and ECPKA
enzymatic assay. We compared the ECPKA EIA with an ECPKA
enzymatic assay that measures antigen (7). The ECPKA enzymatic
assay exhibited a significant overlap between patients with cancer
(n = 66) and normal controls (n = 66) in frequency and mean
values (patients: frequency, 83%; mean value, 130 mU/mL; normal
controls: frequency, 20%; mean value, 60 mU/mL), indicating a lack
of sensitivity and specificity (Fig. 2A). A comparison of individual
anti-ECPKA autoantibody titers obtained by EIA with those
measured by PKA enzymatic assay showed no correlation between
the two assays (Fig. 2B).
The sensitivity and specificity of the autoantibody EIA (Fig. 1)
and those of the ECPKA assay (Fig. 2) were evaluated using ROC
(16) plots (Fig. 3). At this point of interaction, the cutoff value for
anti-ECPKA autoantibody titer was 1.3, exhibiting the highest
sensitivity whereas maintaining a high specificity. The autoantibody
EIA showed 90% sensitivity (95% confidence interval, 0.80-0.92) and
Results
ECPKA autoantibody detection in the sera of patients with
cancer. Our speculation that ECPKA excretion might elicit the
induction of serum autoantibodies led us to develop a novel EIA
method to measure the anti-IgG autoantibody for ECPKA.
We tested the sera of patients with various types of cancer (see
Materials and Methods; n = 295), controls (n = 155), and patients
with diseases other than cancer (n = 55) for the presence of antiECPKA autoantibody using the EIA method. Anti-ECPKA autoantibody titers were expressed arbitrarily as ratios to the mean
absorbance of normal control sera. Compared with the antiECPKA autoantibody titers of normal controls, a >1.3 ratio was
considered positive (Fig. 1).
The titers for anti-ECPKA autoantibody in sera from patients
with cancer and from controls are shown in Fig. 1. Both frequency
and mean titer of the patients with cancer are significantly higher
( frequency, 90%; mean titer, 3.0) than those of the normal controls
( frequency, 12%; mean titer, 1.0).
We also examined whether the level of ECPKA autoantibody is
detectable as a function of tumor type. The results obtained with
different types of tumors were: breast (n = 20; mean titer, 3.05),
colon (n = 24; mean titer, 2.95), lung (n = 6; mean titer,
1.95), melanoma (n = 50; mean titer, 3.00), ovary (n = 20; mean
titer, 2.85), pancreas (n = 6; mean titer, 3.10), prostate (n = 30; mean
titer, 2.95), and renal cell (n = 40; mean titer, 2.58). These results
show that the ECPKA autoantibody is produced, not as a function of
specific tumor type, but by various cancer cell types.
Cancer Res 2006; 66: (18). September 15, 2006
Figure 1. Titers of ECPKA autoantibodies in sera from patients with cancer
with various cancer cell types, patients with disease other than cancer, and
a control group. Anti-ECPKA IgG autoantibodies were measured by a
newly developed EIA (see Materials and Methods). Antibody titers are arbitrarily
expressed as ratios to the mean absorbance of the normal control sera.
Values >1.3 (broken line ) were considered positive. The EIA experiments were
done three or more times with each tumor type with serum samples consisting
of equal numbers of patients and controls with varying total sample sizes of
24, 46, and 92. Various cell types of cancers were included (total, n = 295;
see Materials and Methods). As controls, normal sera (n = 155, from blood
bank) and sera from patients with systemic erythematosus (n = 20), Carney
complex (n = 25), and other diseases (n = 10) were used. The assay was
reproducible, with within-run and between-run coefficients of variation of <0.5%
and <5.8%, respectively. A control serum was included on all plates to
monitor plate-to-plate variation. Variations never exceeded 5%, and values were
therefore not corrected. The differences between groups (patients versus
controls) were calculated to have significance at P < 0.05.
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Autoantibody ECPKA as a Cancer Biomarker
Figure 2. A, ECPKA enzymatic activity in sera from
patients with cancer and from the control group. Values
obtained from patients with cancer and from the control
group were (patients with cancer: frequency, 83%;
mean value, 130 mU/mL; controls: frequency, 21%; mean
value, 60 mU/mL). Values >75 mU/mL (above the
broken line ) were considered positive. B, correlation
between anti-ECPKA autoantibody titer and ECPKA
activity in sera from patients with cancer and controls.
A comparison of individual anti-ECPKA antibody titers and
ECPKA enzymatic activities in sera from patients with
cancer and controls exhibited coefficients of 0.003
for patients with cancer and 0.001 for controls; these are
statistically insignificant.
88% specificity (95% confidence interval, 0.90-0.95) in discriminating between patients with cancer and the control group (Fig. 3). The
enzymatic assay exhibited 83% sensitivity and 80% specificity, with
an area under the curve (AUC) of 0.86938 (compared with the
autoantibody EIA AUC of 0.93675; Fig. 3). These results show that
the ECPKA autoantibody EIA did better than the enzymatic assay in
distinguishing between patients with cancer and controls.
Immunologic identification of ECPKA autoantibody. We
examined the immunologic identification of ECPKA autoantibody
present in the sera of patients with cancer (Fig. 4). Randomly
selected patients’ sera exhibited immuno-cross-reactivity toward
the purified PKA Ca protein (40 kDa; Fig. 4, strips 5-9), whereas
no such immuno-cross-reactivity for Ca protein was observed in
normal sera (Fig. 4, strips 10-14). Importantly, the immuno-crossreactivity for Ca of ECPKA autoantibody in patients’ sera was comparable to those of polyclonal and monoclonal antibodies raised
against the Ca protein (Fig. 4, compare strips 1-4 with strips 5-9).
Autoantibody EIA method to detect other cancer antigens.
To examine whether the autoantibody detection method of the
present study can be extended to other cancer antigens (extracellularly secreted) that produce autoantibodies, an experiment was
done using HCT-15 (a multidrug-resistant human colon carcinoma
grown in nude mice; National Cancer Institute, Frederick, MD)
tumor extracts as a cancer antigen source. These results plotted on a
ROC curve showed that the autoantibody EIA had 92% sensitivity
(95% confidence interval, 0.80-0.95) and 84% specificity (95% confidence interval, 0.78-0.90) in discriminating between patients with
cancer and controls. The observed titers of autoantibodies for cancer
antigens in sera from patients with cancer were shown with different
types of cancer. The results show that it is possible to use the autoantibody detection method described here for ECPKA to detect other
cancer antigens that produce autoantibodies against the antigens.
not to distinguish the stage and site of malignancy. The autoantibody assay was done on sera from active malignancies at various
stages and the same samples were assayed with the conventional
PKA enzymatic assay (antigen assay) for comparison. The results
showed that the autoantibody-EIA assay exhibited better sensitivity
and specificity than did the conventional antigen assay.
The ECPKA autoantibody present in patient’s sera was immunologically cross-reactive with the purified preparation of PKA
catalytic (Ca) subunit and the cross-reactivity was comparable to
that of the polyclonal and monoclonal antibodies raised against
the Ca protein (Fig. 4), demonstrating the specificity of the sera
autoantibody against ECPKA and the immunologic identity of
ECPKA with the Ca subunit of PKA.
The anti-ECPKA autoantibody was elevated almost regardless of
the site or cell types of the malignancy; that is, anti-ECPKA
Discussion
Cancer diagnosis is a serious step in cancer management.
Therefore, we vigorously validated the method and carefully evaluated the data (Fig. 1). We measured the level of autoantibodies
generated against ECPKA, a cancer antigen recently identified
(7, 8), from the growth of various tumors by using serum
samples and found that the accuracy and sensitivity of the present
method exceeds that of the conventional assays. Our purpose was
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Figure 3. ROC plots of ECPKA autoantibody EIA and ECPKA enzymatic assay
in patients with cancer and control sera. The sensitivity and specificity of the
ECPKA autoantibody EIA (from the data of Fig. 1) and those of ECPKA
enzymatic assay (Fig. 2A) are presented in black and red ROC curves,
respectively. The autoantibody EIA had a sensitivity of 90% and specificity of
87% (AUC, 0.936750), and the enzymatic assay showed a sensitivity of 83% and
specificity of 80% (AUC, 0.86938).
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Figure 4. Western blotting of anti-ECPKA autoantibody in sera from
patients with cancer and controls. Lane M, molecular weight marker; lane Ca,
stained with Coomassie blue (1 Ag); strip 1, blotted with PolySC, polyclonal
anti-Ca antibody (Santa Cruz Biotechnology, Santa Cruz, CA); strips 2 to 4,
blotted with monoclonal anti-Ca antibodies, Mab 16, 44, and 72 (Strategic
BioSolutions, Newark, DE); strips 5 to 9, blotted with sera from patients with
cancer (10,000-fold dilution); strips 10 to 14, blotted with normal controls’ sera
(10,000-fold dilution); strips 1 to 14, each contained 1 Ag purified PKA Ca.
autoantibody is a measure of malignant transformation in all cells,
not specific to one type of cancer. Unlike tests such as CEA, which
measures less well-defined antigens and whose serum levels tend
to be inconsistent but elevated late in the disease (3), the antiECPKA autoantibody test measures the autoantibody of a
well-defined cancer antigen, ECPKA, whose serum levels are
specifically up-regulated in the sera of patients with cancer and
are regulated by the changes in the intracellular levels of PKA-I (7).
In the present study, as negative controls of the ECPKA
autoantibody test, we chose sera from patients with Carney complex
(spotty skin pigmentation that can accompany multiple endocrine
neoplasia; ref. 18) and sera from patients with systemic lupus
erythematosus (an autoimmune disease; ref. 17). The absence or low
level of PKA-I is closely/causatively associated with both of these
diseases (17, 18). Our results show that both sera from patients with
Carney complex and lupus erythematosus exhibited ECPKA autoantibody titers as low as that of normal controls (Fig. 1), supporting
the specificity of the ECPKA autoantibody test for cancer diagnosis.
Among the most important criteria for cancer markers is the
ability to distinguish cancer from inflammatory diseases and diseases
other than cancer. Because known cancer markers all depend on the
measurement of cancer antigens, distinguishing between inflammation and cancer is difficult using these cancer markers. In the present
study, we have presented a novel biomarker, autoantibody ECPKA,
that could make it possible to distinguish between cancer, inflammation, and diseases other than cancer. We have shown here that the
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promising, its ultimate performance in multi-institutional studies
must yet be determined.
Acknowledgments
Received 3/21/2006; revised 4/28/2006; accepted 5/26/2006.
Grant support: Intramural Research Program of the NIH, National Cancer
Institute (P.I., Y.S. Cho-Chung), Bethesda, MD.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Jeffrey Schlom (National Cancer Institute, Bethesda) for providing
serum samples. Serum samples from some of the melanoma patients were provided by
the John Wayne Cancer Institute Specimen Repository, which is funded by the
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Correction
Autoantibody ECPKA as a Cancer Biomarker
In the article on autoantibody ECPKA as a cancer biomarker in
the September 15, 2006 issue of Cancer Research (1), the correct
spelling of the seventh author’s name is Islam U. Khan.
1. Nesterova MV, Johnson N, Cheadle C, Bates SE, Mani S, Stratakis CA, Khan IU,
Gupta RK, Cho-Chung YS. Autoantibody cancer biomarker: extracellular protein
kinase A. Cancer Res 2006;66:8971–4.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-66-21-COR
www.aacrjournals.org
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Cancer Res 2006; 66: (21). November 1, 2006
Autoantibody Cancer Biomarker: Extracellular Protein
Kinase A
Maria V. Nesterova, Natalie Johnson, Christopher Cheadle, et al.
Cancer Res 2006;66:8971-8974.
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