Download An Enzyme-linked Immunosorbent Assay for

(CANCER RESEARCH 50, 6229-62.14. October 1. 1990|
An Enzyme-linked Immunosorbent Assay for Cancer Procoagulant and Its Potential
as a New Tumor Marker1
Stuart G. Gordon2 and Barbara A. Cross
Departments of Medicine fS. G. G., B. A. C.] and Pathology fS. G. G.J and University of Colorado Cancer Center fS. G. G./, University of Colorado Health Sciences
Center. Denver, Colorado 80262
ABSTRACT
Cancer procoagulant (CP) is a M, 68,000 cysteine proteinase that
initiates blood coagulation and is expressed by a variety of malignant
cells but not by normally differentiated cells. Polyclonal immunoglobulin
G and monoclonal immunoglobulin M antibodies were developed to
purified CP and used to develop an enzyme-linked immunosorbent assay
to analyze the antigen in human serum samples. The purpose of this
preliminary study was to determine whether or not the analysis of CP in
the serum might be a useful tumor marker.
Pure CP was added to normal serum to establish a quantitative
standard curve; the correlation coefficient of seven standard curves was
0.99. The upper limit of the normal range was established with 46 normal
sera (mean ±2 SD = 0.57 /ig/ml). A total of 128 blinded serum samples
were analyzed: 54 were from cancer patients (29 with gastrointestinal
cancer, 22 with lung cancer, and three with urogenital cancer); 20 were
from benign disease patients; and 54 were from normal individuals. All
of the 13 early stage cancers were >0.57 Mg/ml(positive), 31 of 41 (76%)
of the late stage cancers were positive; overall, 44 of the 54 cancers
(81%) were positive. Forty-nine of 54 (91%) of the normal sera and 16
of 20 (SI)'/ ) of the benign disease sera were negative. Overall, the assay
had a sensitivity of 81% and a specificity of 88%.
INTRODUCTION
Despite many therapeutic advances, early detection of malig
nancy has great potential as a means of affecting outcome and
survival of cancer patients. All too often we find the disease too
far advanced and therapeutic options limited. Earlier diagnosis
and treatment can affect outcome as evidenced by screening
Papanicolou smears for squamous cell carcinoma of the cervix
(1).
The development of an assay with similar potential for early
detection of other malignancies would be of considerable aid to
clinical oncologists. Recently, investigators have identified sev
eral substances that are produced by tumor cells for use as
diagnostic markers of cancer. These include the germ cell
markers human choriogonadotropin and a-fetoprotein, which
are very specific markers, and less sensitive and specific markers
such as CEA,3 CA-12.5, CA-19.9, CA-15.3, PAP, and others.
Recent reviews describe the use of these and other tumor
markers in diagnostic and therapeutic applications (2-6).
During our studies to identify a substance that initiated the
Received 1/23/90; accepted 7/5/90.
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.
1This work was supported by a grant from the National Cancer Institute, NIH,
contracts from Western Biomedicai Development Corp. and E. I. DuPont deNemours & Company, and a gift from L. G. Hickey.
2To whom requests for reprints should be addressed.
3The abbreviations used are: CEA, carcinoembryonic antigen; PAP, prostatic
acid phosphatase; CP, cancer procoagulant; ELISA, enzyme-linked immunosor
bent assay; IgG, immunoglobulin G; IgM, immunoglobulin M; SDS, sodium
dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PAGIF, polyacrylamide gel isoelectric focusing; HPLC, high-pressure liquid chromatography: CV,
coefficient of variation; COPD, chronic obstructive pulmonary disease; GI, gas
trointestinal; GU. gastrourogenital; PBT, 0. l Mphosphate buffer (pH7.4) contain
ing 0.05 % Tween -20.
abnormal blood coagulation associated with malignant disease,
we discovered (7), purified, and partially characterized CP (8,
9). To determine the distribution of CP activity in various
malignant and normal tissues, a variety of extracts of serumfree culture media and cell suspensions of malignant or trans
formed cells as well as their normal counterparts were examined
(10-13). It was found that CP activity existed in the extracts of
malignant cells, but not in extracts from normal tissue or serumfree media from normal cells in tissue culture. Careful enzy
matic and immunological characterization of extracts of malig
nant and benign melanocytic tissue revealed CP in the mela
noma extracts and not in the benign nevi extracts (14). In acute
nonlymphocytic leukemia, CP was identified in most of the
cytological subtypes but not in control mononuclear cells from
either peripheral blood or bone marrow aspirates (15). In ad
dition, a virtually identical procoagulant protein has been pu
rified from human amnion-chorion tissue (fetal origin), imply
ing that CP may be an oncofetal protein (16, 17). These data
were sufficiently suggestive of the unique expression of CP by
malignant cells to warrant the preliminary study to evaluate the
potential of CP as a tumor marker. A double antibody "sand
wich" ELISA was developed and used to assay for CP in the
serum of cancer patients and of noncancerous control individ
uals. These preliminary results suggest that CP has potential
as a tumor marker, and they are sufficiently encouraging to
warrant additional work on the assay and to expand the study.
MATERIALS
AND METHODS
Purification of Cancer Procoagulant Antigen. Cancer procoagulant
was purified from extracts of rabbit V2 carcinoma and human amnionchorion by immuno- and p-chloromercurial benzoate affinity chroma
tography as described previously (9). Briefly, CP was purified by immunoaffinity chromatography in which the immunoaffinity resin was
prepared by coupling purified goat polyclonal IgG to cyanogen bromideactivated Sepharose. Extracts were equilibrated overnight at 5°C,the
column was washed thoroughly with 20 mivi barbila! buffer (pH 7.4),
and bound antigen was eluted with 3 M NaSCN (Sigma, St. Louis,
MO). Trace contaminants were removed by adsorbing CP to an organomercurial resin, washing contaminants off, and eluting CP with 2 HIM
and 5 mM HgCl2. The CP sample was a single band on SDS-PAGE
and PAGIF. It had the enzymatic properties of CP, including procoagulant activity in normal and Factor Vll-deficient human plasma (7,
8); it directly activated human Factor X in a 2-stage assay (8); and its
activity was inhibited by mercury, iodoacetamide, and peptidyl diazomethyl ketone and was reactivated by KCN (8, 9, 18). The purified CP
preparation was used to immunize mice and to screen hybrid cells for
antibody.
Polyclonal Antibodies to Cancer Procoagulant. The polyclonal goat
antibody was raised by standard methodology (9). Briefly, the goat was
immunized and boosted with s.c. injections of between 50 and 100 ^g
of purified enzyme from rabbit V2 carcinoma suspended in Freund's
adjuvant. The goat IgG fraction was partially purified from the serum
by standard purification techniques, including ammonium sulfate fractionation and DEAE-cellulose ion exchange chromatography. A trace
contaminant of an antibody to normal rabbit serum protein was re
moved by coupling normal rabbit serum proteins to cyanogen bromide-
6229
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ENZYME-LINKED
IMMINOSORBENT
activated Sepharose and passing the antibody preparation over this
"normal rabbit serum column." Potential cross-reactivity to normal
human serum proteins was eliminated in the same way, by adsorbing
the antibody preparation to human serum protein-resin. The purified
antibody was tested for cross-reactivity with normal rabbit serum and
normal human serum by crossed immunodiffusion; it was not innminoreactive with either.
Hybridoma Antibodies to Cancer Procoagulant. BALB/c mice
(Charles River) were immunized with cancer procoagulant to induce a
B-cell immune response, according to standard procedures (19, 20).
Briefly, spleen lymphocytes from immunized mice were hybridized with
the P3/X63-AG8.653 variant of mouse myeloma cells with 50% poly
ethylene glycol (21). The hybrids were plated with 2 x 10' normal
mouse spleen cells/ml as a feeder layer, and the unhybridized myeloma
cells were eliminated by hypoxanthine aminopterin thymidine suicide.
Cells from antibody-positive wells were expanded and cloned at 0.1 to
0.5 cells per well with a feeder layer of normal spleen cells. Antibodies
from positive wells were classified using specific subclass anti-mouse
immunoglobulins according to the technical information provided in
the Zymed Lab Mono Ab-ID EIA kit (22). Antibodies were screened
by a standard single antibody microtiter plate immunoassay using
purified CP absorbed to microtiter wells. Alkaline phosphatase-labeled
anti-mouse IglVlantibody was used as the signal antibody, and the color
was read at 405 nm on a Dynatech microtiter plate reader.
Antibody Production and Purification. The anti-CP IgM was produced
in the ascites of Pristane-treated BALB/c mice (23). During the analysis
of immunoglobulin in the ascites fluid, procoagulant activity was dis
covered in the cell-free ascites. This procoagulant had the characteristics
ascribed to cancer procoagulant, including inhibition by mercury, ini
tiation of coagulation of Factor VII-depleted human plasma, and immunoprecipitation with the polyclonal goat antibody (9). To effectively
utilize the antibody in immunoassays or other immunological experi
ments, it was necessary to separate the antigen from the antibody. This
was accomplished by making the ascites 6 M urea and running the
sample over a 1.5-m agarose (Biorad, Richmond, CA) column (1.5 x
60 cm) that was preequilibrated in 6 M urea. The fractions containing
the IgM protein peak were pooled, dialyzed against 3 changes of 20
imi barbila! buffered saline (pH 7.4), and concentrated over an Amicon
PM-50 membrane.
An alternative procedure used to separate antigen from the IgM was
to make the ascites 6% Polyethylene Glycol 6000 to precipitate the
IgM. The precipitate was recovered by centrifugation. decanting the
supernatant, and redissolving it in a minimal volume (about I ml/10
ml of ascites) of 0.1 M phosphate-buffered saline (pH 7.4). The solubilized IgM was made 50% with ethylene glycol and stored at 5°Cuntil
it was further purified by HPLC (Waters, Inc.). A semipreparative GF450-XL (DuPont) was preequilibrated in 0.1 M sodium phosphate
buffer in 50% ethylene glycol, up to 2 ml of the antibody preparation
were applied and eluted at 1 ml/min, and fractions were collected to
separate IgM from the antigen.
The purified IgM was concentrated on an Amicon PM-50 ultrafiltration membrane, made 50% with glycerol, and stored at —20°C
until it
ASSAY FOR CANCER PROCOAGULANT
Each step of the assay was checked to determine the optimum
conditions for the assay. The dilution of monoclonal antibody was
checked from 1:1 dilution to 1:50,000 dilution. Human serum albumin
(0.1%) was as good a blocking agent as if not better than, the same
concentration of bovine serum albumin, ovalbumin, normal goat serum,
normal human serum, and normal rabbit serum. Higher (to 5%) and
lower (to 0.01%) concentrations of each of the protein solutions were
tested without improving the assay results. The time (1, 2, 4, and 16 h)
and temperature (5°,25°,and 37°C)for coating and blocking the wells
were determined as well as each incubation. A variety of washing buffers
and concentrations of nonionic detergents were tested to determine the
most effective way to remove substances that increased nonspecific
binding. Thus, each of the conditions was carefully checked to arrive at
the sequence of steps that provided the best assay system.
Standard Curve and Calibration for the Assay. A standard curve was
prepared by adding known amounts of pure CP to normal serum.
Concentrations from 0 to 30 Mg/ml were used. Each microtiter plate
contained a standard curve, 6 to 8 normal control sera and a high and
a medium control pooled sera, and the unknown serum samples.
Analysis of Human Serum Samples. The initial serum samples in
cluded 46 normal volunteers from the Health Sciences Center that were
processed to establish the normal range. The remaining samples were
blinded and analyzed without knowing the patient's identity or diag
nosis. After the assay was complete, the results were analyzed, and the
samples were identified and compared with the results of the analysis.
In the 128 serum samples that were analyzed in this blinded fashion,
there were 54 sera from cancer patients, 54 from normal individuals,
and 20 from individuals with benign disease (Table 1). The samples
were usually analyzed in triplicate in at least 3 different assays.
A small group of samples were collected in duplicate or triplicate to
determine the effect of anticoagulants and hemolysis on the assay.
Heparin, citrate, and NaF plasma samples had essentially the same CP
values as did their serum counterparts, but the results had greater
variability. EDTA-plasma samples had assay results that were about
50% lower than their serum counterparts and greater variability. Sam
ples with slight hemolysis had comparable results to unhemolyzed
serum, but extensive hemolysis resulted in high analysis results. Lipemic serum had high analysis results, but the addition of clearing
agents, such as Liposolve, provided samples that appeared to have
reliable assay values.
RESULTS
The identification of procoagulant activity in the ascites
indicated the need to purify the antigen from the antibody.
Ascites was made 6 M urea to dissociate antigen-antibody
1100
you
2 600 60
was used in an assay. Testing of the ascites IgM antibody and the
purified IgM antibody clearly demonstrated that the purified IgM had
greater immunoreactivity, as expected.
Development of a Double Antibody Immunoassay. The wells of an
luminimi II 96-well microtiter plate were coated with 0.19 ^g/well of
monoclonal antibody (1:80 dilution of stock IgM) for 2 h at 25°C,and
«700
'S
£
„500
emptied and washed 3 times with PBT, and 50 ¿il
(0.26 nM) of alkaline
phosphatase-conjugated goat anti-CP antibody (24) in phosphate-buff
ered saline were added and incubated for l h at 37°C.Finally the pnitrophenyl phosphate substrate was added, incubated at 37°Cfor
400 40 «
200 20°
U
the plate was washed 3 times with PBT. Open sites in the well were
blocked with 0.1% normal human serum albumin in O.I M phosphate
buffer (pH 7.6) and stored in a vacuum dessicator at 5°Cuntil it was
used in an assay. Serum (25 ^1) was diluted in an equal volume of PBT
In the microtiter wells and incubated for 2 h at 5°C.The wells were
<
300
9 17 25 33 41 49 57 65
Fraction Number
Fig. 1. An elution profile from a I.S-m agarose chromatography column
showing the separation of hybridoma IgM antibody from CP antigen. Four ml of
ascites were made 6 mol with crystalline urea, applied to the column that was
preequilibrated in 6 M urea, and eluted with 6 M urea in 20 HIMbarbital buffer.
Protein was monitored by measuring absorbance at 280 nm (
). Fractions
were dialy/cd against 20 HIMbarbital buffer, and IgM was measured in a microtiter
plate assay using alkaline phosphatase-labcled goat anti-mouse IgM antibody
(
). The clotting activity was measured with the recalcification clotting time
of citrated normal human plasma (
).
between 30 and 60 min, and read on a microtiter plate reader at 405
nm.
6230
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ENZYME-LINKKD
IMMl NOSORBKN I ASSAY FOR CANTER PRCX'OAGULANT
complex. The two proteins were then physically separated on a
1.5-m agarose gel filtration column (Fig. 1) with an exclusion
limit of 1.5 x IO7 daltons which can separate M, 850,000 IgM
from M, 68,000 cancer procoagulant. The HPLC Chromato
graphie profile of the GF-450-XL purification of IgM was
essentially the same as that in Fig. 1; using ethylene glycol as
the dissociating agent provided IgM with better stability and
immunoreactivity, so this procedure was adopted as the method
of choice. The purified IgM fractions were assayed for CP by
(a) crossed immunodiffusion against polyclonal anti-CP goat
antibody, (b) assaying the sample for iodoacetamide sensitivity
and Factor VII-independent procoagulant activity, and (c) as
saying for IgM-CP complex in a modified double antibody
immunoassay by determining the reactivity of the purified IgM
(containing IgM-CP) with the goat antibody in the microtiter
wells and SDS-PAGE.
Assay Characteristics. Each sample was assayed in triplicate
on a microtiter plate, and the CV for a representative 56 sets
of triplicate wells was 7.9 ±6.0%.
The CV for 44 duplicate samples analyzed in the same assay
was 8.6 ±5.9%. The interassay CV for 35 samples that were
assayed in from 2 to 5 different assays was 13.2 ±9.4%. There
were 7 pairs of samples that were blinded with different code
numbers and integrated into the pool of samples; the mean CV
was 18.1 ±5.8%. Hand pipetting of all steps in the assay
probably contributed to the elevated CVs in the assay.
A standard curve was prepared from the samples of normal
serum that contained known amounts of CP. This standard
curve was used to establish linearity of the assay, to quantitate
the level of CP in the unknown serum samples, and to determine
the reproducibility of the assay. Fig. 2 shows the mean ±
standard error of the mean of 7 standard curves. The regression
analysis of the data shows a correlation coefficient of 0.99. The
results of each plate were analyzed by checking the linearity of
the standards.
The mean and one standard deviation of the 46 normal serum
samples was 0.36 i¿g/m\±0.11 Mg/ml. Thus, the mean + 2
standard deviations was used to establish the normal range of
0 to 0.57 ¿ig/ml;results above 0.57 Mg/ml were considered
positive.
Analysis of Blinded Samples. A total of 54 normal samples
from volunteers were "blinded" with code numbers and inte
grated into the pool of 54 blinded cancer patient samples and
20 benign disease patient samples. Of the 54 normal sera, 49
(91%) were negative, while 16 of 20 benign disease sera were
negative. In the overall noncancer population, 65 of the 74 sera
(88%) were negative; i.e., the results were <0.57 ¿ig/ml.
Table 1 shows some of the clinical details of the 54 cancer
patients and 20 benign disease patients from whom serum
samples were obtained. Fig. 3 shows the distribution of data
for each of the groups of samples. In the 29 samples from
patients with gastrointestinal cancers, all of which were coloTable 1 List of clinical profiles of patients that provided blood for the blinded
serum samples analy:ed in the study, including information about the patient 's
type and stage of cancer or the patient's type of benign disease and the number of
samples in each group
There were 54 cancer patient sera, 20 benign disease sera, and 54 normal sera
analyzed in the study: there were 46 nonblinded normal control scrum samples
used to establish the "normal range": they are not included in the tabulation of
the results.
Stage
III
Malignancies
Gastrointestinal
Colorectal adenocarcinoma
Colorectal carcinoma
Lung
Squamous cell
Small cell carcinoma
Urogenital
Prostate
Ovarian
2
IV
All
25
28
7
15
1
2
Benign disease
Lung
Asthma
Pneumonia
coro
4
(1 patient also had BPH)°
3
6
5
(1 patient also had asbestosis)
Emphysema
Other
Inflammatory bowel
I
Deep vein thrombosis
' BPH. benign prostatic hypertrophy.
3422
•Gl
85cJO3
480
r2 =O.99
91%••
4a'54 =
80%•
& GU
78%••...
25-32 =
19/22 -86%X•
28(u
O>«-~
~^§- 2
380
.£
° °>1 71
i1'71i_
280
•"•
••X•
^-^
0)o
Ift
O
*,,c
<o
0n
180
.•
x"•
•
i-14
0x
•*
•rtfS&fBENIGN1670
•
."
••
••* ^
;••
' •'
80 •
••••
0
•X
<**x•
*
•.*.
•.•LUNG•
.NORMAL
-20
eo
180
270
360
450
ng 0.1 ml Cancer Procoagulant
Fig. 2. The mean of seven standard curves in which pure CP antigen was
added to normal human serum and analyzed by the ELISA procedure described
in the text. The blank signal ut the normal serum was subtracted from each
sample to calibrate the signal for each level of antigen. Each standard was assayed
in triplicate on each microtiter plate, as described in the text. These standards
were used to quantitate the level of antigen in the normal and blinded serum
samples. The correlation coefficient was 0.99, and the equation of the leastsquares fit line was y = -9.43 + 7.69.V.Points, mean; bars. SEM.
Fig. 3. Analysis of 128 cancer and noncancer sera, the results of the blinded
samples analyzed with the ¡mmunoassay.The normal + 2 SDs (0.57 fig/ml) was
used as the upper limit of normal. The samples with CP levels less than 0.57 >ig/
ml were considered normal, and those above that level were considered positive.
The left column shows the 54 normal serum samples that were assayed, and
49 of them were normal. The second column from the left shows the results of 20
benign disease patient sera: 16 of them were normal. An ,Vin the benign disease
group is a patient with asbestosis and COPD. who may have cancer. The third
column from the left shows 29 gastrointestinal cancer patient sera and 3 urogenital
cancer patient sera; 22 of IheGI samples and all of the GU samples were positive.
Three gray tone dots with white centers represent the GU cancer samples. The last
column on the right shows 22 lung cancer patient serum samples; 19 of them were
positive. A' represents the early stage (Stage I and II) cancers.
6231
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ENZYME-LINKED IMMUNOSORBENT ASSAY FOR CANCER PROCOAGULANT
rectal cancers, there were 22 (76%) positive (>0.57 Mg/ml) sera.
There were 22 serum samples from patients with lung tumors,
7 with squamous carcinoma and IS with small cell carcinoma.
Nineteen of these sample (86%) were positive.
All 3 sera from patients with urogenital cancer were positive.
Table 2 shows the data analyzed by stage of disease. All of
the Stage A and B gastrointestinal cancer patients. Stage I and
II lung cancer patients, and the patient with Stage II ovarian
carcinoma were positive. Thus, 13 of 13 sera (100%) from early
stage cancers were positive. In contrast, only 19 of 27 (73%) of
the later stage gastrointestinal cancer patient sera and 10 of 13
(77%) of the Stage III and IV lung cancer patient sera were
positive. Both of the late stage urogenital cancer patient sera
were positive.
There were 20 serum samples from patients with benign
disease; 18 of them had various types of pulmonary disease.
Two patients had more than one type of benign disease; one
patient with asthma also had benign prostatic hypertrophy. A
second patient with COPD had a diagnosis of asbestosis and
was a 2-pack/day smoker; this was one of the 4 benign disease
patients that tested positive in the assay. A second positive
benign disease patient had severe COPD and was also a heavy
cigarette smoker.
Statistical analysis of the results (25) indicates that the ability
of the ELISA to correctly identify patients with cancer (i.e., the
sensitivity of the test) was 76% for colorectal tumors, 86% for
lung tumors, and 100% for the urogenital malignancies, with
an overall sensitivity of 81%. The sensitivity for early stage
cancers was 100%. The specificity of the test (the percentage of
noncancer patient sera that were negative) was 88%.
antigens that have been identified to date are also present in
sera from some normal individuals and from a spectrum of
benign disease conditions, making them inadequate for accept
able detection or screening procedures. However, they have
been clinically useful for monitoring cancer therapy; most no
table among these are CEA and «-fetoprotein. Recently the list
of tumor markers has grown substantially with the addition of
PAP, tissue polypeptide antigen, pancreatic oncofetal antigen,
CA-19.9, CA-12.5, CA 15.3, and others. There are recent
reviews of tumor markers and tumor-associated antigens (2-6).
Many of these markers have not been fully tested or widely
accepted for clinical use. The list of "current tumor markers"
changes frequently, and few tumor antigens hold the place of
prominence and security that CEA and a-fetoprotein have held
for the past decade.
Carcinoembryonic antigen, first described by Gold and Freedman (26) and by Fuks and Gold (27), is a high-molecular-weight
(M, 180,000 to 200,000) glycoprotein that is present in various
types of malignant disease, including tumors of the GI tract
(30%), stomach (72%), pancreas (88%), breast (24%), and res
piratory tract (30%) (28). Elevated plasma CEA is associated
with a variety of nonmalignant disorders, such as chronic
obstructive pulmonary disease (57%) and most inflammatory
diseases, including pneumonia (46%) and hepatitis (40%). CEA
is elevated in about 10% and 15% of normal nonsmokers and
smokers, respectively.
a-Fetoprotein is a M, 70,000 protein (29, 30) that is elevated
in the serum of about 80% of patients with hepatic tumors,
almost all those with teratocarcinomas (31, 32).
CP was originally found during studies to identify a substance
that initiated the abnormal blood coagulation associated with
malignant disease. Abnormalities of the coagulation system
DISCUSSION
(33-35), including disseminated intravascular coagulation (36,
It is widely believed that malignant cells produce substances
37), have been repeatedly identified as pathological features of
that are not produced by normal cells, but finding and identi
malignant disease. Furthermore, fibrin is deposited in and
fying these substances have been very difficult. With the enor
around solid tumors (38-42), is associated with blood-borne
mous medical promise and human effort devoted to the quest
malignant cells (43, 44), and is thought to participate in various
for reliable tumor markers, it is not surprising that many tumor
aspects of the malignant process (45-50).
antigens have been identified and partially characterized during
These documented abnormalities in the coagulation system
the past 20 yr in the hope of providing methods for early
in malignant disease led investigators to look for procoagulants
detection and therapeutic monitoring of cancer. However, many
in neoplastic tissue (51-54). We identified, purified to homo
substances that are found in cancer cells are also found in some
geneity, and partially characterized CP, a unique cysteine pronormal and noncancerous (benign) diseases. Thus, many of the teinase (7-9, 55) that initiates coagulation by directly activating
Factor X in the coagulation cascade. CP is a single polypeptide
Table 2 Summary of the results of the study
protein with a molecular weight of 68,000 and an isoelectric
"Early" stage cancers refer to Stage I and II cancers, and "late" stage cancers
point of 4.8. It is rich in serine and glycine and contains no
refer to Stage III and IV cancers. The number correctly identified is in the
numerator, and the total number of samples analyzed is in the denominator. For
measurable carbohydrate (9). Its physical, chemical, and enzy
"all" cancers samples, correct identification means a positive result (>0.57 ¿ig/
matic properties are different than other coagulation enzymes
ml), and for all "noncancer" samples, correct identification means a negative
(9).
result (<0.57 ¿ig/ml).Calculations of the sensitivity (the ability to correctly
identify patients with cancer) and specificity (the ability to correctly identify
During the course of these studies, it became important to
individuals that do not have cancer) are derived from these results. These values
establish
whether or not CP was found in association with
test the efficiency of this test in accurately identifying people with cancer from
normal tissues (11) and cells (10, 12); it was found in neither
those that are free of disease.
intact normal cells, extracts, nor culture media. Furthermore,
a procoagulant protein was purified from human amnion-chorion tissue (of fetal origin), and its properties are virtually
Cancer patients
identical, with the exception of minor differences in amino acid
(76)
Gl tumors
10/13(77)
19/22(86)
Lung tumors
9/9(100)
composition, to CP from rabbit V2 carcinoma (16, 17), B16
1/1 (100)
2/2(100)
3/3(100)
GU tumors
melanoma, and all the human tumor extracts that have been
TotalNoncancer
13/13(100)49/54(91)31/41 (76)All22/2944/54(81)
analyzed. This suggests that the procoagulant protein is highly
patients
conserved across species lines and tumor types and that CP
Normal
may be an oncofetal protein.
16/20(80)
Benign
TotalEarly3/3(100)° 65/74 (88)StageLate19/26(73)
During the development of the hybridoma antibody, we had
°Numbers in parentheses, percentage of samples correctly identified.
trouble obtaining consistent results with the screening assay
6232
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ENZYME-LINKED
IMMIINOSORBENT
ASSAY FOR CANCER PROCOAGULANT
and getting an IgM with adequate immunoreactivity from ascites samples that contained from 5 to 7 ¿/gof IgM/ml of
ascites. The ascites contained a procoagulant that had all the
enzymatic properties of CP (8, 9) and cross-reacted with antiCP polyclonal goat antibody on an Ouchterlony immunodiffusion plate (9). Subsequently, it found that the tissue culture
medium from both the parent P3/X63-AG8.653 cell line and
other hybrid cell clones from colleagues in the Health Sciences
Center contained CP with immunoreactive and enzymatic iden
tity. Although unexpected, it was logical since both the parent
cell line and the hybrid cell lines have the malignant phenotype
and should produce the CP antigen if CP is an oncofetal protein
as suggested above.
It was necessary to separate the antigen and IgM so that the
IgM could be used in an immunoassay. We chose to take
advantage of the difference in the physical size of the two
molecules for the separation. Two methods were used to dis
sociate CP from IgM: 6 M urea and 50% ethylene glycol; both
methods facilitated dissociation so the IgM could be resolved
from the CP on gel filtration chromatography (1.5-m agarose
and GF-450 HPLC). However, IgM purification in 50% ethyl
ene glycol resulted in more stable IgM that was free of CP.
In this preliminary study, antibodies to CP were developed
and used in an immunoassay to measure CP in serum from
cancer and noncancer patients. The assay correctly identified
44 or 81% of the 54 serum samples from cancer patients.
Ninety-one % of the normal sera and 80% of the benign disease
sera were negative; 88% of the 74 noncancer sera were correctly
identified. The mean + 2 standard deviations seems to be a
reasonable estimator of the 90th percentile of the sample dis
tribution (the limit excludes the upper 10% of the observations).
Thus, the percentage of false-positive results for the normal
sera is within the limits of the expected value.
In a previous pilot study (56), 140 blinded serum samples
were assayed from patients with colorectal (26), lung (27),
breast (35), prostate (6), lymphoma (6), osteosarcoma (4), and
gall bladder cancer (3) and from 70 normal individuals and 10
patients with benign disease. Approximately 90% of the sam
ples were correctly identified. Thus the results of both studies
are similar and support the hypothesis that CP may be a tumor
marker.
Comparison of these data with data from an extensive 1974
clinical trial of CEA (57) shows that CEA had a sensitivity of
72% for colorectal cancer, 91 % for pancreatic cancer, 74% for
lung cancer, and 47% for breast cancer (58). CEA had low
specificity (ranging from 30% to 82%) due to the large number
of benign conditions that led to positive serum CEA levels. In
contrast, CP had a sensitivity of 76% for colorectal cancer and
86% for lung cancers. The specificity of the CP assay was 88%.
In addition, the assay correctly identified 100% of the early
stage cancers. These statistics compare favorably with CEA.
Thus, the data support the hypothesis that CP may be an
oncofetal protein with the distribution necessary to be an effec
tive tumor marker.
There was a substantial percentage of false-negative cancer
patient sera in both the gastrointestinal and lung cancer groups
of patients with late stage cancer. It was noted that several of
these "false negative" patients were terminal at the time the
blood samples were obtained and that they died from their
malignancy shortly thereafter. We examined the blood samples
for endogenous anti-CP antibodies because such antibodies in
the serum of these cancer patients would probably interfere
with the immunoassay. These serum samples were reacted with
pure CP on Ouchterlony-crossed immunodiffusion, and 3 of
the 4 showed immunoprecipitation bands, suggesting the pres
ence of endogenous antibodies. Alternatively, there is evidence
for a decreased production of CP activity in the serum of rats
with late stage epithelioma (59), suggesting that CP production
in late stage cancers may decrease. In the false negative samples,
CP may be associated with a serum protein or other substance
produced by the tumor that binds CP, blocks the epitope, and
inhibits the immunoreactivity of CP in the assay. The false
negative results in late stage cancer will be the subject of
continued studies.
The objective of this study was to develop a basic immunoas
say system and to obtain adequate preliminary evidence from
blinded cancer and noncancer sera to test the concept that CP
may be a useful tumor marker. Although we concluded that the
immunoassay for CP had reasonable clinical potential as a
useful tumor marker test, there were several difficulties that
need to be resolved by refinements of the assay. These include
the following: (a) developing a more stable monoclonal IgG
hybridoma antibody to replace the IgM antibody used in the
assay; (b) establishing a method to screen serum samples for
endogenous anti-CP antibody; (c) doing follow-up studies on
the normal and benign disease populations if they have high
CP levels in their blood to determine whether or not they have
malignant disease; and (<•/)
studying a large number of benign
diseases to develop a list of noncancerous conditions associated
with elevated serum CP levels.
These are questions that cannot be answered in the present
study but will be examined very closely in the next phase of
evaluation of CP as a tumor marker. In spite of these problems,
we feel that the immunoassay for CP in serum may have
reasonable clinical potential as a cancer diagnostic or monitor
ing test for cancer.
REFERENCES
1. Lunt, R. Worldwide early detection of cervical cancer. Obstet. Gynecol.. 63:
708-713. 1984.
2. Pohl. A. L., Francesconi, M., Ganzinger, U. C., Graninger, W., Lenzhofer,
R. S.. and Moser, K. V. Present value of tumor markers in the clinic. Cancer
Detect. Prevent., 6: 7-20, 1983.
3. Klavins, J. V. Advances in biological markers for cancer. Ann. Clin. Lab.
Sci.. 13: 275-280. 1983.
4. Sulitzeanu, D. Human cancer-associated antigens: present status and impli
cations for immunodiagnosis. Adv. Cancer Res., 44: 1-42, 1985.
5. Virji, M. A., Mercer, D. W., and Herberman, R. B. Tumor markers in cancer
diagnosis and prognosis. CA. 38: 105-126, 1988.
6. Sikorska. II.. Schuster. J., and Gold. P. Clinical application of carcinoembryonic antigen. Cancer Detect. Prevent.. 12: 321-355. 1988.
7. Gordon. S. G., Franks, J. J., and Lewis, B. Cancer procoagulant A: a factor
X-activating procoagulant from malignant tissue. Thrombos. Res., 6: 127137, 1975.
8. Gordon, S. G., and Cross, B. A. A factor X-activating cysteine protease from
malignant tissue. J. Clin. Invest., 67: 1665-1671. 1981.
9. Falanga, A., and Gordon, S. G. Isolation and characterization of cancer
procoagulant: a cysteine proteinase from malignant tissue. Biochemistry, 24:
5558-5567. 1985.
10. Gordon. S. G.. and Lewis, B. Comparison of procoagulanl from normal and
transformed fibroblasts. Cancer Res., 38: 2467-2472. 1978.
11. Gordon. S. G., Franks. J. J.. and Lewis. B. J. Comparison of procoagulants
in extracts of normal and malignant human tissue. J. Nati. Cancer Inst., 62:
773-776. 1979.
12. Gordon, S. G., Gilbert, L. C., and Lewis, B. J. Analysis of procoagulant
activity of intact cells from tissue culture. Thrombos. Res., 26: 379-387,
1982.
13. Gordon. S. G., and Benson, B. A. Analysis of serum cancer procoagulant
activity and its possible use as a tumor marker. Thrombos. Res., 56: 431440. 1989.
14. Donati. M. B., Passerini, C. G., Casali, B., Falanga. A.. Vannotti, P., Fossati,
G.. Semeraro, V. and Gordon. S. G. Cancer procoagulant in human tumor
cells: evidence from melanoma patients. Cancer Res., 46: 6471-6474, 1986.
15. Falanga. A.. Alessio. A. G., Donati. M. B., and Barbui, T. A new procoagulant
in acute leukemia. Blood. 71: 870-875, 1988.
6233
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ENZYME-LINKED IMMUNOSORBENT ASSAY FOR CANCER PROCOAGULANT
16. Gordon. S. G., Hashiba, U.. Cross, B. A.. Poole, M. A., and Falanga. A. A
cysteine proteinase procoagulant from amnion-chorion. Blood. 66: 12611265, 1985.
17. Falanga. A., and Gordon, S. G. Comparison of properties of cancer procoagulant and human amnion-chorion procoagulant. Biochim. Biophys. Acta,
831: 161-165, 1985.
18. Falanga, A.. Shaw, I .. Donati, M. B., Consonni, R., Barbui. T.. and Gordon.
S. G. Inhibition of cancer procoagulant by peptidyl diazomethyl ketones and
pcptidyl sulfonium salts. Thrombos. Res., 54: 389-398, 1989.
19. Köhler.G., and Milstein. C. Derivation of specific antibody-producing tissue
culture and tumor lines by cell fusion. Eur. J. Immunol.. 6: 511-519, 1976.
20. Yelton. D. E., Margulies. D. H.. Diamond. B.. and Scharff. M. D. Plasmacytomas and hybridomas. Development and applications. In: R. H. Kennet!,
T. J. McKearn, and K. B. Bechtol (eds.). Monoclonal Antibodies, pp. 3-17.
New York: Plenum Press. 1980.
21. Kohler. G., and Milstein, C. Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature (Lond.). 256:495-497. 1975.
22. Technical Bulletin, pp. 12 to 13. San Francisco, CA: Zymed Laboratory.
Inc.. 1984.
23. Johnslone. A., and Thorpe. R. Immunochemistry in Practice, pp. 27-40.
Boston: Blackwell Scientific Publications. 1982.
24. Engvali, E. Enzyme immunoassay ELISA and EMIT. Methods Enzymol.,
70:419-439, 1980.
25. Archer, P. G. The predictive value of clinical laboratory test results. Am. J.
Clin. Pathol., 69: 32-35, 1978.
26. Gold. P.. and Freedman, S. O. Demonstration of tumor-specific antigens in
human colonie carcinomata by immunological tolerance and absorption
techniques. J. Exp. Med., 121: 439-462. 1965.
27. Fuks. A., and Gold, P. Carcinoembryonic antigen. J. Tumor Oncol., I: 111,1986.
28. Beatty. J. D., and Terz. J. J. Value of carcinoembryonic antigen in clinical
medicine. Prog. Clin. Cancer, 8: 9-29, 1982.
29. Alpen, E., Drysdale. J. W., Isselbacher, K. J., and Schur. P. H. Human
alpha-fetoprotein. J. Biol. Chem., 247: 3792-3798, 1972.
30. Watabe. H. Purification and chemical characterization of alpha-fetoprotein
from rat and mouse. Int. J. Cancer. 13: 377-388. 1974.
31. Ruddon. R. W. Tumor markers in the recognition and management of poorly
differentiated neoplasms and cancers of unknown primary. Semin. Oncol.,
9: 416-426, 1982.
32. Mclntire. K. R.. Waldmann. T. A.. Moertel, C. G.. and Go, V. L. W. Serum
a-fetoprotein in patients with neoplasms of the gastrointestinal tract. Cancer
Res., 35: 991-996, 1975.
33. Miller, S. P., Sanchez-Avalos, J.. Stefanski, T., and Zuckerman, L. Coagu
lation disorders in cancer. I. Clinical and laboratory studies. Cancer (Phila.),
20:1452-1465, 1967.
34. Pochedly, C., Miller. S. P.. and Mehta. A. Hypercoagulable state in children
in acute leukemia or disseminated solid tumors. Oncology. 28: 517-522,
1973.
35. Rickles. F. R.. and Edwards. R. L. Activation of blood coagulation in cancer:
Trousseau's syndrome revisited. Blood. 62: 14-31, 1983.
36. Gore, J. M.. Appelbaum, J. S.. Greene. H. L.. Dexter. L.. and Dalen. J. E.
Occult cancer in patients with acute pulmonary embolism. Ann. Intern.
Med., 96: 556-560, 1982.
37. Rasche. H., and Dietrich. M. Hemostatic abnormalities associated with
malignant disease. Eur. J. Cancer, 13: 1053-1064, 1977.
38. Franks. J. J.. Gordon, S. G., Kao. B., Sullivan. T., and Kirch. D. Increased
fibrin formation with tumors and its genesis. In: R. Bianchi. G. Mariani. and
A. S. McFarlane (eds.). Plasma Protein Turnover, pp. 423-440. London:
Macmillan Press, 1976.
39. Ogura, T., Tsubura, E., and Yamamura, Y. Localization of radioiodinated
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
fibrinogen in invaded and metastasized tumor tissue of Walker carcinosarcoma. Gann. 61: 443-449. 1970.
Dvorak, H. F., Dvorak, A. M.. Manseau. E. J.. Wilberg, L., and Churchill.
W. H. Fibrin gel investment associated with line 1 and line 10 solid tumor
growth, angiogenesis. and fibroplasia in guinea pigs. Role of cellular immu
nity, myofibroblasts. microvascular damage, and infarction in line 1 tumor
regression. J. Nati. Cancer Inst., 62: 1459-1477. 1979.
O'Meara. R. A. Q., and Jackson, R. D. Cytological observations on carci
noma. Irish J. Med. Sci., 391: 327-328, 1958.
Day, E. D., Planinsek, J. A., and Pressman, D. Localization in viro of
radioiodinated anti-rat-fibrin antibodies and radioiodinated rat fibrinogen in
the Murphy rat lymphosarcoma and in other transplantable rat tumors. J.
Nati. Cancer Inst., 22: 413-426, 1959.
Donati. M. B.. Poggi. A., and Semeraro, N. Coagulation and malignancy. In:
L. Poller (eds.). Recent Advances in Blood Coagulation, pp. 227-259. Edin
burgh: Churchill-Livingstone, 1981.
Wood, S., Jr. Experimental studies of the intravascular dissemination of
ascitic V2 carcinoma cells in the rabbit with special reference to fibrinogen
and fibrinolytic agents. Bull Swiss Acad. Med. Sci., 20: 92-121. 1964.
Kinjo. M. Lodgement and extravasation of tumour cells in blood-borne
metastasis: an electron microscope study. Br. J. Cancer, 38: 293-301, 1978.
Millar, R. C., and Ketchem, A. S. The effect of heparin and warfarin on
primary and metastatic tumors. J. Med., 5: 23-31, 1974.
Hilgard, P.. and Thornes. R. D. Anticoagulants in the treatment of cancer.
Eur. J. Cancer. 12: 755-762. 1976.
Skolnik, G.. Alpsten. M., and Ivarsson. L. Studies on mechanisms involved
in metastasis formation from circulating tumor cells. J. Cancer Res. Clin.
Oncol., 97: 249-256, 1980.
Colucci, M., Delaini, F., Vitti, G., Locati, D., Poggi, A., Semeraro, N., and
Donati, M. B. Cancer cell procoagulant activity, warfarin, and experimental
métastases,¡n:K. Hellman. P. Hilgard. and S. Eccles (eds.). Metastasis:
Clinical and Experimental Aspects. Martinus Nijhoff, 1980.
Donati, M. B.. Poggi, A., Mussoni, L., de Gaetano, G., and Garattini. S. In:
S. B. Day. ci al. (eds.). Hemostasis and experimental cancer dissemination.
Cancer Invasion and Metastasis: Biologic Mechanisms and Therapy, pp.
151-160. New York: Raven Press. 1977.
Pineo. G. F.. Rogoeczi. E., Hatton, M. W. C., and Brian. M. C. The activation
of coagulation by extracts of mucus: a possible pathway of intravascular
coagulation accompanying adenocarcinomas. J. Lab. Clin. Med., 82: 255266. 1973.
Boggust. W. A.. O'Brien. D. J.. O'Meara, R. A. W.. and Thornes, R. D. The
coagulative factors of normal human and human cancer tissue. Irish J. Med.
Sci., 477: 131-144, 1967.
Dvorak, H. F., Quay, S. C., Orenstein, N. S., et al. Tumor shedding and
coagulation. Science (Wash. DC). 212: 923-924. 1981.
Hilgard. P., and Whur, P. Factor X-activating activity from Lewis lung
carcinoma. Br. J. Cancer. 41:642-643, 1980.
Gordon. S. G. A proteolytic procoagulant associated with malignant trans
formation. J. Histochem. Cytochem.. 29: 457-463, 1981.
Gordon, S. G. Cancer procoagulant. In: Proceedings of Koloman Laki
Memorial Symposium on Hemostasis in Cancer, pp. 19-32. Boca Raton.
FL: CRC Press. 1987.
Hansen. H. I.. Snyder. J. J.. Miller. E. Vandevoorde. J. P.. Miller, O. N.,
Hiñes.L. R.. and Burns. J. J. Carcinoembryonic antigen (CEA) assay. Human
Pathol., 5: 139-147. 1974.
Galen, R. S., and Gambino, S. R. Beyond Normality: The Predictive Value
and Efficiency of Medical Diagnosis, pp. 76-80. New York: J. Wiley & Son.
1975.
Meilicki. W.. and Wierbicki, R. Cancer procoagulant in serum of rats during
development of experimental epitheloma. Int. J. Cancer, 45: 125-126, 1990.
6234
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
An Enzyme-linked Immunosorbent Assay for Cancer
Procoagulant and Its Potential as a New Tumor Marker
Stuart G. Gordon and Barbara A. Cross
Cancer Res 1990;50:6229-6234.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/50/19/6229
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.