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THIS IS VOLUME 1 OF 2
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1
Use of protein SAHH as a marker for colorectal cancer
The present invention relates to the diagnosis of colorectal cancer. It
discloses the
use of the protein SAHH (= S-adenosylhomocysteine hydrolase) in the diagnosis
of
colorectal cancer. Furthermore, it especially relates to a method for
diagnosis of
colorectal cancer from a stool sample, derived from an individual by measuring
SAHH in said sample: Measurement of SAHH can, e.g., be used in the early
detection or diagnosis of colorectal cancer.
Cancer remains a major public health challenge despite progress in detection
and
therapy. Amongst the various types of cancer, colorectal cancer (= CRC) is one
of
the most frequent cancers in the Western world.
The earlier cancer can be detected/diagnosed, the better is the overall
survival rate.
This is especially true for CRC. The prognosis in advanced stages of tumor is
poor.
More than one third of the patients will die from progressive disease within
five
years after diagnosis, corresponding to a survival rate of about 40% for five
years.
Current treatment is only curing a fraction of the patients and clearly has
the best
effect on those patients diagnosed in an early stage of disease.
With regard to CRC as a public health problem, it is essential that more
effective
screening and preventative measures for colorectal cancer be developed.
The earliest detection procedures available at present for colorectal cancer
involve
using tests for fecal blood or endoscopic procedures. However, significant
tumor
size must typically exist before fecal blood is detected. With regard to
detection of
CRC from a stool sample, the current state of the art is the guaiac-based
fecal occult
blood test.
In the recent years a tremendous amount of so-called colon specific or even so-
called colorectal cancer specific genes has been reported. The vast majority
of the
corresponding research papers or patent applications are based on data
obtained by
analysis of RNA expression patterns in colon (cancer) tissue versus a
different tissue
or an adjacent normal tissue, respectively. Such approaches may be summarized
as
differential mRNA display techniques.
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As an example for data available from mRNA-display techniques, WO 01/96390
shall be mentioned arid discussed. This application describes and claims more
than
two hundred isolated polynucleotides and the corresponding polypeptides as
such,
as well as their use in the detection of CRC. However, it is general knowledge
that
differences on the level of mRNA are not mirrored by the level of the
corresponding
proteins. A protein encoded by a rare mRNA may be found in very high amounts
and a protein encoded by an abundant rnRNA may nonetheless be hard to detect
and find at all. This lack of correlation between mRNA-level and protein level
is
due to reasons like mRNA stability, efficiency of translation, stability of
the protein,
Z O etc.
There also are recent approaches investigating the differences in protein
patterns
between different tissues or between healthy and diseased tissue in order to
identify
candidate marker molecules which might be used in the diagnosis of CRC.
Briinagel, G., et al., Cancer Research 62 (2002) 2437-2442, have identified
seven
nuclear matrix proteins which appear to be more abundant in CRC tissue as
compared to adjacent normal tissue. No data from liquid and stool samples
obtained from an individual are reported.
WO 02/078636 reports about nine colorectal cancer-associated spots as found by
surface-enhanced laser desorption and ionization (SELDI). These spots are seen
more frequently in sera obtained from patients with CRC as compared to sera
obtained from healthy controls. However, the identity of the molecules)
comprised
in such spot, e.g., its (their sequence), is not known.
Despite the large and ever growing list of candidate protein markers in the
field of
CRC, to date clinical/diagnostic utility of these molecules is not known. In
order to
be of clinical utility a new diagnostic marker as a single marker should be at
least as
good as the best single marker known in the art. Or, a new marker should lead
to a
progress in diagnostic sensitivity and/or specificity either if used alone or
in
combination with one or more other markers, respectively. The diagnostic
sensitivity and/or specificity of a test is best assessed by its receiver-
operating
characteristics, which will be described in detail below.
At present, for example, diagnostic blood tests based on the detection of
carcinoembryonic antigen (CEA), a tumor-associated glycoprotein, are available
to
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assist diagnosis in the field of CRC. CEA is increased in 95% of tissue
samples
obtained from patients with colorectal, gastric, and pancreatic cancers and in
the
majority of breast, lung, and head and neck carcinomas (Goldenberg, D.M., et
al., J.
Natl. Cancer Inst. (Bethesda) 57 (1976) 11-22). Elevated CEA levels have also
been
reported in patients with nonmalignant disease, and many patients with
colorectal
cancer have normal CEA levels in the serum, especially during the early stage
of the
disease (Carriquiry, L.A., and Pineyro, A., Dis. Colon Rectum 42 (1999) 921-
929;
Herrera, M.A., et al., Ann. Surg. 183 ( 1976) 5-9; Wanebo, H.J., et al., N.
Engl. J.
Med. 299 (1978) 448-451). The utility of CEA as measured from serum or plasma
in detecting recurrences is reportedly controversial and has yet to be widely
applied
(Martell, R.E., et al., Int. J. Biol. Markers 13 (1998) 145-149; Moertel,
C.G., et al.,
JAMA 270 ( 1993) 943-947).
In light of the available data, serum CEA determination possesses neither the
sensitivity nor the specificity to enable its use as a screening test for
colorectal
cancer in the asymptomatic population (Reynoso, G., et al., JAMA 220 (1972)
361-
365; Sturgeon, C., Clinical Chemistry 48 (2002) 1151-1159).
Samples taken from stool have the advantage that such sampling is easily
possible
by non-invasive means.
As mentioned above, the guaiac test is currently most widely used as a
screening
assay for CRC from stool. The guaiac test, however, has both poor sensitivity
as well
as poor specificity. The sensitivity of the guaiac-based fecal occult blood
tests is
~26%, which means 74% of patients with malignant lesions will remain
undetected
(Ahlquist, D.A., Gastroenterol. Clin. North Am. 26 ( 1997) 41-55). The
visualization
of precancerous and cancerous lesions represents the best approach to early
detection, but colonoscopy is invasive with significant costs, risks, and
complications (Silvis, S.E., et al., JAMA 235 ( 1976) 928-930; Geenen, J.E.,
et al.,
Am. J. Dig. Dis. 20 ( 1975) 231-235; Anderson, W.F., et al., J. Natl. Cancer
Institute
94 (2002) 1126-1133).
The sensitivity and specificity of diagnostic alternatives to the guaiac test
have been
recently investigated by Sieg, A., et al., Int. J. Colorectal Dis. 14 ( 1999)
267-271.
Especially the measurement of hemoglobin and of the hemoglobin-haptoglobin
complex from stool specimen have been compared. It has been noted that the
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hemoglobin assay has an unsatisfactory sensitivity for the detection of
colorectal
neoplasms. Whereas cancer in its progressed carcinoma stage is detected with a
sensitivity of about 87% the earlier tumor stages are not detected with a
sufficient
sensitivity. The hemoglobin-haptoglobin complex assay was more sensitive in
the
detection of earlier stages of CRC. This more sensitive detection was
accompanied
by a poor specificity. Since poor specificity, however, translates to a high
number of
unnecessary secondary investigations, like colonoscopy, an assay with a poor
accuracy also does not meet the requirements of a generally accepted screening
assay.
A further alternative method to the guaiac test for detection of CRC in stool
has
been published recently and consists in the detection of the colorectal cancer-
specific antigen, "minichromosome maintenance protein 2" (MCM2) by
immunohistochemistry in colonic cells shed into stool. Due to the small study
size,
conclusion on the diagnostic value for detection of colorectal cancer is
preliminary.
However, the test seems to have only limited sensitivity to detect right-sided
colon
cancer. (Davies, R.J., et al., Lancet 359 (2002) 1917-1919).
The identification of an early CRC tumor marker that would allow reliable
cancer
detection or provide early prognostic information by non-invasive means from a
stool specimen could lead to a diagnostic assay that would greatly aid in the
diagnosis and in the management of this disease. Therefore, an urgent clinical
need
exists to improve the diagnosis of CRC, especially from stool. It is
especially
important to improve the early diagnosis of CRC, since for patients diagnosed
early
on chances of survival are much higher as compared to those diagnosed at a
progressed stage of disease.
It was the task of the present invention to investigate whether a new marker
can be
identified which may aid in CRC diagnosis. Preferably such marker would be
present in stool and allow for a non-invasive diagnosis.
Surprisingly, it has been found that use of protein SAHH can at least
partially
overcome the problems known from the state of the art.
The present invention therefore relates to a method for the diagnosis of
colorectal
cancer comprising the steps of a) providing a stool sample obtained from an
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individual, b) contacting said sample with a specific binding agent for SAHH
under
conditions appropriate for formation of a complex between said binding agent
and
SAHH, and c) correlating the amount of complex formed in (b) to the diagnosis
of
colorectal cancer.
In a preferred embodiment the stool sample is processed to obtain a processed
sample liquid which is more convenient to handle than a stool specimen. Such
processed sample is then incubated with the specific binding agent for SAHH.
The
present invention therefore also relates to a method for the diagnosis of
colorectal
cancer comprising the steps of a) providing a stool sample obtained from an
individual, b) processing said sample to obtain a processed liquid sample, c)
contacting said processed liquid sample with a specific binding agent for SAHH
under conditions appropriate for formation of a complex between said binding
agent and SAHH, and d) correlating the amount of complex formed in (c) to the
diagnosis of colorectal cancer. A preferred method uses a stool sample
obtained
from an individual.
Another preferred embodiment of the invention is a method for the diagnosis of
colorectal cancer comprising the steps of a) processing a stool sample
obtained
from an individual to obtain a processed liquid sample b) contacting said
processed
liquid sample with a specific binding agent for SAHH under conditions
appropriate
for formation of a complex between said binding agent and SAHH, and c)
correlating the amount of complex formed in (b) to the diagnosis of colorectal
cancer.
In another preferred embodiment the stool sample is processed to retrieve
colonycytes which are then smeared on a microscopic slide. Such processed
sample
is then incubated with the specific binding agent for SAHH. The present
invention
therefore also relates to a method for the diagnosis of colorectal cancer
comprising
the steps of a) providing a stool sample obtained from an individual, b)
processing
said sample to retrieve colonycytes, c) contacting said processed sample with
a
specific binding agent for SAHH under conditions appropriate for formation of
a
complex between said binding agent and SAHH, and d) correlating the amount of
complex formed in (c) to the diagnosis of colorectal cancer.
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The protein SAHH (S-adenosylhomocysteine hydrolase; SWISS-PROT: P23526) is
characterized by the sequence given SEQ ID NO: 1. The corresponding cloned
human cDNA encodes for a 48-kDa protein which catalyzes the following
rerversible reaction: S-adenosyl-L-homocysteine + H2O ~ adenosine + L-
homocysteine (Cantoni, G.L., Annu. Rev. Biochem. 44 (1975) 435-451).
Hershfield,
M.S., and Francke, U., Science 216 (1982) 739-742, located the corresponding
gene
to chromosome 20 and later on Coulter-Karis, D.E., and Hershfield, M.S., Ann.
Hum. Genet. 53 ( 1989) 169-175, sequenced the full-length cDNA. Recently, the
structure of SAHH has been resolved (Turner, M.A., et al., Cell. Biochem.
Biophys.
33 (2000) 101-125).
S-adenosyl-L-homocysteine is formed by the donation of a methyl group of S-
adenosylmethionine, a universal methyl donor, to a methyl acceptor (e.g. DNA).
Subsequently, it is hydrolyzed to adenosine and L-homocysteine by SAHH.
Inhibition of SAHH results in accumulation of cellular S-adenosyl-L-
homocysteine,
which acts as feedback inhibitor on most methylation reactions (Chiang, P.K.,
Pharmacol. Ther. 77 (1998) 115-134). Since methylation of nucleic acids and
phospholipids is crucial in cellular growth, differentiation and
carcinogenesis
(Chiang, P.K., et al., FASEB J. 10 (1996) 471-480; Cui, Z., et al., J. Biol.
Chem. 269
( 1994) 24531-24533; Szyf, M., et al., Pharmacol. Ther. 70 ( 1996) 1-37),
inhibitors of
SAHH are an interesting pharmacological target. Consequently, there are a
number
of inhibitors of SAHH described as reviewed in Chiang, P.K., Pharmacol. Ther.
77
(1998) 115-134.
A deficiency in SAHH is one of the different causes of hypermethioninemia, a
disease associated with failure to thrive, mental and motor retardation,
facial
dysmorphism, and myocardiopathy (Labrune, P., et al., J. Pediatr. 117 ( 1990)
220-
226).
Immunoblotting techniques were used to determine SAHH levels in hepatic
tissues
(Bethin, K.E., et al., J. Biol. Chem. 270 ( 1995) 20698-20702).
As obvious to the skilled artisan, the present invention shall not be
construed to be
limited to the full-length protein SAHH of SEQ ID NO:1. Physiological or
artificial
fragments of SAHH, secondary modifications of SAHH, as well as allelic
variants of
SAHH are also encompassed by the present invention. Artificial fragments
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preferably encompass a peptide produced synthetically or by recombinant
techniques, which at least comprises one epitope of diagnostic interest
consisting of
at least 6 contiguous amino acids as derived from the sequence disclosed in
SEQ ID
NO:1. Such fragment may advantageously be used for generation of antibodies or
as a standard in an immunoassay. More preferred the artificial fragment
comprises
at least two epitopes of interest appropriate for setting up a sandwich
immunoassay.
In preferred embodiments, the novel marker SAHH may be used for monitoring as
well as for screening purposes. Its use for screening purposes is most
preferred.
When used in patient monitoring the diagnostic method according to the present
invention may help to assess tumor load, efficacy of treatment and tumor
recurrence in the follow-up of patients. Increased levels of SAHH are directly
correlated to tumor burden. After chemotherapy a short term (few hours to 14
days) increase in SAHH may serve as an indicator of tumor cell death. In the
follow-up of patients (from 3 months to 10 years) an increase of SAHH can be
used
as an indicator for tumor recurrence in the colorectum.
In a preferred embodiment the diagnostic method according to the present
invention is used for screening purposes. Le., it is used to assess subjects
without a
prior diagnosis of CRC by measuring the level of SAHH in a stool sample and
correlating the level measured to the presence or absence of CRC.
Colorectal cancer most frequently progresses from adenomas (polyps) to
malignant
carcinomas. The different stages of CRC used to be classified according to
Dukes'
stages A to D.
The staging of cancer is the classification of the disease in terms of extent,
progression, and severity. It groups cancer patients so that generalizations
can be
made about prognosis and the choice of therapy.
Today, the TNM system is the most widely used classification of the anatomical
extent of cancer. It represents an internationally accepted, uniform staging
system.
There are three basic variables: T (the extent of the primary tumor), N (the
status of
regional lymph nodes) and M (the presence or absence of distant metastases).
The
TNM criteria are published by the UICC (International Union Against Cancer),
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_g_
Sobin, L.H., Wittekind, Ch. (eds): TNM Classification of Malignant Tumours,
fifth
edition, 1997.
What is especially important is, that early diagnosis of CRC translates to a
much
better prognosis. Malignant tumors of the colorectum arise from benign tumors,
i.e. from adenoma. Therefore, best prognosis have those patients diagnosed at
the
adenoma stage. Patients diagnosed as early as in stage Tis, N0, MO or T1-3;
N0; M0,
if treated properly have a more than 90% chance of survival 5 years after
diagnosis
as compared to a 5-years survival rate of only 10% for patients diagnosed when
distant metastases are already present.
In the sense of the present invention early diagnosis of CRC refers to a
diagnosis at
a pre-malignant state (adenoma) or at a tumor stage where no metastases at all
(neither proximal nor distal), i.e., adenoma, T;S, N0, MO or T1-4; N0; MO are
present. T;S denotes carcinoma iii situ.
In a preferred embodiment the detection of SAHH is used to diagnose CRC as
early
as in the adenoma stage.
It is further preferred, that CRC is diagnosed when it has not yet fully grown
through the bowel wall and thus neither the visceral peritoneum is perforated
nor
other organs or structures are invaded, i.e., that diagnosis is made at stage
T;S, N0,
MO or T1-3; N0; MO (=T;~-3; N0; MO).
The diagnostic method according to the present invention is based on a stool
sample which is derived from an individual. The stool sample is extracted and
SAHH is specifically measured from this processed stool sample by use of a
specific
binding agent.
A specific binding agent is, e.g., a receptor for SAHH, a lectin binding to
SAHH or
an antibody to SAHH. A specific binding agent has at least an affinity of 10'
1/mol
for its corresponding target molecule. The specific binding agent preferably
has an
affinity of 108 1/mol or even more preferred of 1091/mol for its target
molecule. As
the skilled artisan will appreciate the term specific is used to indicate that
other
biomolecules present in the sample do not significantly bind to with the
binding
agent specific for SAHH. Preferably, the level of binding to a biomolecule
other
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than the target molecule results in a binding affinity which is only 10%, more
preferably only 5% of the affinity of the target molecule or less. A most
preferred
specific binding agent will fulfill both the above minimum criteria for
affinity as
well as for specificity.
A specific binding agent preferably is an antibody reactive with SAHH. The
term
antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of
such
antibodies, as well as to genetic constructs comprising the binding domain of
an
antibody.
Any antibody fragment retaining the above criteria of a specific binding agent
can
be used. Antibodies are generated by state of the art procedures, e.g., as
described in
Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 ( 1990)
the
whole book, especially pages 43-78; Elsevier, Amsterdam). In addition, the
skilled
artisan is well aware of methods based on immunosorbents that can be used for
the
specific isolation of antibodies. By these means the quality of polyclonal
antibodies
and hence their performance in immunoassays can be enhanced. (Tijssen, P.,
supra,
pages 108-115).
For the achievements as disclosed in the present invention polyclonal
antibodies
raised in rabbits have been used. However, clearly also polyclonal antibodies
from
different species , e.g. rats or guinea pigs, as well as monoclonal antibodies
can also
be used. Since monoclonal antibodies can be produced in any amount required
with constant properties, they represent ideal tools in development of an
assay for
clinical routine. The generation and use of monoclonal antibodies to SAHH in a
method according to the present invention is yet another preferred embodiment.
As the skilled artisan will appreciate now, that SAHH has been identified as a
marker which is useful in the diagnosis of CRC, alternative ways may be used
to
reach a result comparable to the achievements of the present invention. For
example, alternative strategies to generate antibodies may be used. Such
strategies
comprise amongst others the use of synthetic peptides, representing an epitope
of
SAHH for immunization. Alternatively, DNA Immunization also known as DNA
vaccination may be used.
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For measurement, the stool sample is obtained from an individual. An aliquot
of
the stool sample may be used directly. Preferably an aliquot of the stool
sample is
processed to yield a liquid sample.
The stool sample is preferably used or processed directly after sampling or
stored
cooled or more conveniently stored frozen. Frozen stool samples can be
processed
by thawing, followed by dilution in an appropriate buffer, mixing and
centrifugation. Supernatants are used as liquid sample for subsequent
measurement
of marker SAHH.
An aliquot of the processed stool sample is incubated with the specific
binding
agent for SAHH under conditions appropriate for formation of a binding agent
SAHH-complex. Such conditions need not be specified, since the skilled artisan
without any inventive effort can easily identify such appropriate incubation
conditions.
As a final step according to the method disclosed in the present invention the
amount of complex is measured and correlated to the diagnosis of CRC. As the
skilled artisan will appreciate there are numerous methods to measure the
amount
of specific binding agent SAHH-complex all described in detail in relevant
textbooks (c~, e.g., Tijssen P., supra, or Diamandis, E.P., et al., eds.
(1996)
Immunoassay, Academic Press, Boston).
Preferably SAHH is detected in a sandwich type assay format. In such assay a
first
specific binding agent is used to capture SAHH on the one side and a second
specific binding agent, which is labeled to be directly or indirectly
detectable is used
on the other side.
As mentioned above, it has surprisingly been found that SAHH can be measured
from a stool sample obtained from an individual sample. No tissue and no
biopsy
sample is required to apply the marker SAHH in the diagnosis of CRC.
Whereas application of routine proteomics methods to tissue samples, leads to
the
identification of many potential marker candidates for the tissue selected,
the
inventors of the present invention have surprisingly been able to detect
protein
SAHH in a stool sample. Even more surprising they have been able to
demonstrate
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that the presence of SAHH in such stool sample obtained from an individual can
be
correlated to the diagnosis of colorectal cancer.
Antibodies to SAHH with great advantage can also be used in established
procedures, e.g., to detect colorectal cancer cells in situ, in biopsies, or
in
immunohistological procedures.
Preferably, an antibody to SAHH is used in a qualitative (SAHH present or
absent)
or quantitative (SAHH amount is determined) immunoassay.
Measuring the level of protein SAHH has proven very advantageous in the field
of
CRC. Therefore, in a further preferred embodiment, the present invention
relates
to use of protein SAHH as a marker molecule in the diagnosis of colorectal
cancer
from a stool sample obtained from an individual.
The term marker molecule is used to indicate that an increased level of the
analyte
SAHH as measured from a bodily fluid or especially a processed stool sample
obtained from an individual marks the presence of CRC.
It is especially preferred to use the novel marker SAHH in the early diagnosis
of
colorectal cancer.
The use of protein SAHH itself, represents a significant progress to the
challenging
field of CRC diagnosis from stool. Combining measurements of SAHH with other
known markers, like hemoglobin or the hemoglobin-haptoglobin complex, or with
other markers of CRC yet to be discovered, leads to further improvements.
Therefore in a further preferred embodiment the present invention relates to
the
use of SAHH as a marker molecule for colorectal cancer in combination with one
or more other marker molecules for colorectal cancer in the diagnosis of
colorectal
cancer from a stool sample obtained from an individual. Preferred selected
other
CRC markers with which the measurement of SAHH may be combined are
hemoglobin and/or the hemoglobin-haptoglobin complex. Very preferred the
marker SAHH is used in combination with hemoglobin. Also very preferred the
marker SAHH is used in combination with the hemoglobin-haptoglobin complex.
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Diagnostic reagents in the field of specific binding assays, like
immunoassays,
usually are best provided in the form of a kit, which comprises the specific
binding
agent and the auxiliary reagents required to perform the assay. The present
invention therefore also relates to an immunological kit comprising at least
one
specific binding agent for SAHH and auxiliary reagents for measurement of
SAHH.
Accuracy of a test is best described by its receiver-operating characteristics
(ROC)
(see especially Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561-
577).
The ROC graph is a plot of all of the sensitivity/specificity pairs resulting
from
continuously varying the decision thresh-hold over the entire range of data
observed.
The clinical performance of a laboratory test depends on its diagnostic
accuracy, or
the ability to correctly classify subjects into clinically relevant subgroups.
Diagnostic
accuracy measures the test's ability to correctly distinguish two different
conditions
of the subjects investigated. Such conditions are for example health and
disease or
benign versus malignant disease.
In each case, the ROC plot depicts the overlap between the two distributions
by
plotting the sensitivity versus 1 - specificity for the complete range of
decision
thresholds. On the y-axis is sensitivity, or the true-positive fraction
[defined as
(number of true-positive test results) (number of true-positive + number of
false-
negative test results)]. This has also been referred to as positivity in the
presence of
a disease or condition. It is calculated solely from the affected subgroup. On
the x-
axis is the false-positive fraction, or 1 - specificity [defined as (number of
false-
positive results) / (number of true-negative + number of false-positive
results)]. It
is an index of specificity and is calculated entirely from the unaffected
subgroup.
Because the true- and false-positive fractions are calculated entirely
separately, by
using the test results from two different subgroups, the ROC plot is
independent of
the prevalence of disease in the sample. Each point on the ROC plot represents
a
sensitivity/-specificity pair corresponding to a particular decision
threshold. A test
with perfect discrimination (no overlap in the two distributions of results)
has an
ROC plot that passes through the upper left corner, where the true-positive
fraction
is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0
(perfect
specificity). The theoretical plot for a test with no discrimination
(identical
distributions of results for the two groups) is a 45° diagonal line
from the lower left
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corner to the upper right corner. Most plots fall. in between these two
extremes. (If
the ROC plot falls completely below the 45° diagonal, this is easily
remedied by
reversing the criterion for "positivity" from "greater than" to "less than" or
vice
versa.) Qualitatively, the closer the plot is to the upper left corner, the
higher the
overall accuracy of the test.
One convenient goal to quantify the diagnostic accuracy of a laboratory test
is to
express its performance by a single number. The most common global measure is
the area under the ROC plot. By convention, this area is always > 0.5 (if it
is not,
one can reverse the decision rule to make it so). Values range between 1.0
(perfect
separation of the test values of the two groups) and 0.5 (no apparent
distributional
difference between the two groups of test values). The area does not depend
only on
a particular portion of the plot such as the point closest to the diagonal or
the
sensitivity at 90% specificity, but on the entire plot. This is a
quantitative,
descriptive expression of how close the ROC plot is to the perfect one (area =
1.0).
Clinical utility of the novel marker SAHH has been assessed in comparison to
and
in combination with the established marker hemoglobin using a receiver
operator
curve analysis (ROC; Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561-
577). This analysis has been based on well-defined patient cohorts consisting
of 30
samples each from patients in T1-3; N0; M0, more progressed tumor, i.e., T4
and/or various severity of metastasis (N+ and/or M+), and healthy controls,
respectively.
The following examples, references, sequence listing and figures are provided
to aid
the understanding of the present invention, the true scope of which is set
forth in
the appended claims. It is understood that modifications can be made in the
procedures set forth without departing from the spirit of the invention.
Description of the Figures
Figure 1 Figure 1 shows a typical example of a 2D-gel, loaded with a tumor
sample (left side), and a gel, loaded with a matched control
sample (right side) obtained from adjacent healthy mucosa. The
molecular weight inferred from the position of the protein spot in
the gel is about 48 kDa, the isoelectric point is at about pH 6
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(less). The circle in the enlarged section of these gels indicates the
position for the protein SAHH. This protein was not detectable by
the same method in healthy mucosa.
Figure 2 Typical example of a Western-Blot. A polyacrylamide gel was
loaded with tissue lysates from colorectal tumor tissue and
adjacent healthy control tissue from 3 patients (subject 4: colon ca
(carcinoma), Dukes C; subject 7: colon ca, Dukes C; and subject
13: colon ca, Dukes B) and after electrophoresis the proteins were
blotted onto a nitrocellulose membrane. Presence of SAHH in the
samples was tested using a polyclonal rabbit anti-SAHH serum.
Lanes containing tumor lysates are indicated with "T", lanes
containing normal control tissue with "N". The arrow indicates
the position in the gel of the SAHH band. All tumor samples give
a strong signal at the position of SAHH, whereas only a weale
signal can be detected in the lysates from adjacent normal control
tissue.
Abbreviations
ABTS 2,2'-Azino-di- [3-ethylbenzthiazoline sulfonate
(6)]
diammonium salt
BSA bovine serum albumin
cDNA complementary DNA
CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-
1-propane-
sulfonate)
DMSO dimethyl sulfoxide
DTT dithiothreitol
EDTA ethylene diamine tetraacetic acid
ELISA enzyme-linked immunosorbent assay
HRP horseradish peroxidase
IAA iodoacetamid
IgG immunoglobulin G
IEF isoelectric focussing
IPG immobilized pH gradient
LDS lithium dodecyl sulfate
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MALDI-TOF matrix-assisted laser desorption/ionisation-time of flight
mass spectrometry
MES mesityl, 2,4,6-trimethylphenyl
OD optical density
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PI isoelectric point
RTS rapid translation system
SDS sodium dodecyl sulfate
Example 1
Identification of SAHH as a potential colorectal cancer marker
Sources of tissue
In order to identify tumor-specific proteins as potential diagnostic markers
for
colorectal cancer, analysis of three different kinds of tissue using
proteomics
methods is performed.
In total, tissue specimen from 10 patients suffering from colorectal cancer
are
analyzed. From each patient three different tissue types are collected from
therapeutic resections: tumor tissue (> 80% tumor) (T), adjacent healthy
tissue (N)
and stripped mucosa from adjacent healthy mucosa (M). The latter two tissue
types
serve as matched healthy control samples. Tissues are immediately snap frozen
after
resection and stored at - 80°C before processing. Tumors are diagnosed
by
histopathological criteria.
Tissue preparation
0.8-1.2 g of frozen tissue are put into a mortar and completely frozen by
liquid
nitrogen. The tissue is pulverized in the mortar, dissolved in the 10-fold
volume
(w/v) of lysis buffer (40 mM Na-citrate, 5 mM MgCl2, 1% Genapol X-080, 0.02%
Na-azide, Complete~ EDTA-free [Roche Diagnostics GmbH, Mannheim,
Germany, Cat. No. 1 873 580] ) and subsequently homogenized in a Wheaton~
glass
homogenizer (20 x loose fitting, 20 x tight fitting). 3 ml of the homogenate
are
subjected to a sucrose-density centrifugation (10-60% sucrose) for 1 h at 4500
x g.
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After this centrifugation step three fractions are obtained. The fraction on
top of
the gradient contains the soluble proteins and is used for further analysis.
Isoelectric focussing (IEF) and SDS-PAGE
For IEF, 3 ml of the suspension are mixed with 12 ml sample buffer (7 M urea,
2 M
thiourea, 2% CHAPS, 0.4% IPG buffer pH 4-7, 0.5% DTT) and incubated for 1 h.
The samples are concentrated in an Amicon° Ultra-15 device
(Millipore GmbH,
Schwalbach, Germany) and the protein concentration is determined using the Bio-
Rad~ protein assay (Cat.No. 500-0006; Bio-Rad Laboratories GmbH, Miinchen,
Germany) following the instructions of the supplier's manual. To a volume
corresponding to 1.5 mg of protein sample buffer is added to a final volume of
350 ~tl. This solution is used to rehydrate IPG strips pH 4-7 (Amersham
Biosciences, Freiburg, Germany) overnight. The IEF is performed using the
following gradient protocol: 1.) 1 minute to 500 V; 2.) 2 h to 3,500 V; 3.) 22
h at
constant 3,500 V giving rise to 82 kVh. After IEF, strips are stored at -
80°C or
directly used for SDS-PAGE.
Prior to SDS-PAGE the strips are incubated in equilibration buffer (6 M urea,
50 mM Tris/HCI, pH 8.8, 30% glycerol, 2% SDS), for reduction DDT (15 min, +
50 mg DTT/10 ml), and for alkylation IAA (15 min, + 235 mg iodacetamide/10 ml)
is added. The strips are put on 12.5% polyacrylamide gels and subjected to
electrophoresis at 1 W/gel for 1 h and thereafter at 17 W/gel. Subsequently,
the gels
are fixed (50% methanol, 10% acetate) and stained overnight with NoveX M
Colloidal Blue Staining Kit (Invitrogen, Karlsruhe, Germany, Cat No. LC6025,
45-
7101).
Detection of SAHH as a potential marker for colorectal cancer
Each patient is analyzed separately by image analysis with the ProteomeWeaver~
software (Definiens AG, Germany, Miinchen). In addition, all spots of the gel
are
excised by a picking robot and the proteins present in the spots are
identified by
MALDI-TOF mass spectrometry (UltraflexTM Tof/Tof, Bruker Daltonik GmbH,
Bremen, Germany). For each patient, 4 gels from the tumor sample are compared
with 4 gels each from adjacent normal and stripped mucosa tissue and analyzed
for
distinctive spots corresponding to differentially expressed proteins. By this
means,
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protein SAHH is found to be specifically expressed or strongly overexpressed
in
tumor tissue and not detectable or less strongly expressed in healthy control
tissue.
It therefore - amongst many other proteins - qualifies as a candidate marker
for use
in the diagnosis of colorectal cancer.
Example 2
Generation of antibodies to the colorectal cancer marker protein SAHH
Polyclonal antibody to the colorectal cancer marker protein SAHH is generated
for
further use of the antibody in the measurement of serum and plasma and blood
and stool levels of SAHH by immunodetection assays, e.g. Western Blotting and
ELISA.
Recombinant protein expression in E. coli
In order to generate antibodies to SAHH, recombinant expression of the protein
is
performed for obtaining immunogens. The expression is done applying a
combination of the RTS 100 expression system and E.coli. In a first step, the
DNA
sequence is analyzed and recommendations for high yield cDNA silent mutational
variants and respective PCR-primer sequences are obtained using the
"ProteoExpert RTS E.coli HY" system. This is a commercial web based service
(www.proteoexpert.com). Using the recommended primer pairs, the "RTS 100 E.
coli Linear Template Generation Set, His-tag" (Roche Diagnostics GmbH,
Mannheim, Germany, Cat.No. 3186237) system to generate linear PCR templates
from the cDNA and for in-vitro transcription and expression of the nucleotide
sequence coding for the SAHH protein is used. For Western-blot detection and
later purification, the expressed protein contains a His-tag. The best
expressing
variant is identified. All steps from PCR to expression and detection are
carried out
according to the instructions of the manufacturer. The respective PCR product,
containing all necessary T7 regulatory regions (promoter, ribosomal binding
site
and T7 terminator) is cloned into the pBAD TOPO° vector (Invitrogen,
Karlsruhe,
Germany, Cat. No. K 4300/01) following the manufacturer's instructions. For
expression using the T7 regulatory sequences, the construct is transformed
into E.
coli BL 21 (DE 3) (Studier, F.W., et al., Methods Enzymol. 185 (1990) 60-89)
and
the transformed bacteria are cultivated in a 11 batch for protein expression.
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Purification of His-SAHH fusion protein is done following standard procedures
on
a Ni-chelate column. Briefly, 11 of bacteria culture containing the expression
vector
for the His-SAHH fusion protein is pelleted by centrifugation. The cell pellet
is
resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidium
chloride,
imidazole and thioglycerole, followed by homogenization using a Ultra-Turrax
~t .
Insoluble material is pelleted by high speed centrifugation and the
supernatant is
applied to a Ni-chelate chromatographic column. The column is washed with
several bed volumes of lysis buffer followed by washes with buffer, containing
phosphate, pH 8.0 and Urea. Finally, bound antigen is eluted using a phosphate
buffer containing SDS under acid conditions.
Production of monoclonal antibodies against the SAHH
a) Immunization of mice
12 week old A/J mice are initially immunized intraperitoneally with 100 ~g
SAHH.
This is followed after 6 weeks by two further intraperitoneal immunizations at
monthly intervals. In this process each mouse is administered 100 ~.g SAHH
adsorbed to aluminum hydroxide and 109 germs of Bordetella pertussis.
Subsequently the last two immunizations are carried out intravenously on the
3rd
and 2nd day before fusion using 100 ~g SAHH in PBS buffer for each.
b) Fusion and cloning
Spleen cells of the mice immunized according to a) are fused with myeloma
cells
according to Galfre, G., and Milstein, C., Methods in Enzymology 73 ( 1981 ) 3-
46.
In this process ca. 1*10$ spleen cells of the immunized mouse are mixed with
2x10'
myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at
300 x g and 4°C.). The cells are then washed once with RPMI 1640 medium
without
fetal calf serum (FCS) and centrifuged again at 400 x g in a 50 ml conical
tube. The
supernatant is discarded, the cell sediment is gently loosened by tapping, 1
ml PEG
(molecular weight 4000, Merck, Darmstadt) is added and mixed by pipetting.
After
1 min in a water-bath at 37°C., 5 ml RPMI 1640 without FCS is added
drop-wise at
room temperature within a period of 4-5 min. Afterwards 5 ml RPMI 1640
containing 10% FCS is added drop-wise within ca. 1 min, mixed thoroughly,
filled
to 50 ml with medium (RPMI 1640+10% FCS) and subsequently centrifuged for
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min at 400 x g and 4°C. The sedimented cells are taken up in RPMI 1640
medium containing 10% FCS and sown in hypoxanthine-azaserine selection
medium (100 mmol/1 hypoxanthine, 1 ~.g/ml azaserine in RPMI 1640+10% FCS).
Interleukin 6 at 100 U/ml is added to the medium as a growth factor.
5 After ca. 10 days the primary cultures are tested for specific antibody.
SAHH-
positive primary cultures are cloned in 96-well cell culture plates by means
of a
fluorescence activated cell sorter. In this process again interleukin 6 at 100
U/ml is
added to the medium as a growth additive.
c) Immunoglobulin isolation from the cell culture supernatants
10 The hybridoma cells obtained are sown at a density of 1x105 cells per ml in
RPMI
1640 medium containing 10% FCS and proliferated for 7 days in a fermenter
(Thermodux Co., Wertheim/Main, Model MCS-104XL, Order No. 144-050). On
average concentrations of 100 ~.g monoclonal antibody per ml are obtained in
the
culture supernatant. Purification of this antibody from the culture
supernatant is
carried out by conventional methods in protein chemistry (e.g. according to
Bruclc,
C., et al., Methods in Enzymology 121 (1986) 587-695).
Generation of polyclonal antibodies
a) Immunization
For immunization, a fresh emulsion of the protein solution ( 100 ~g/ml protein
SAHH) and complete Freund's adjuvant at the ratio of 1:1 is prepared. Each
rabbit
is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90.
Blood is
drawn and resulting anti-SAHH serum used for further experiments as described
in
examples 3 and 4.
b) Purification of IgG (immunoglobulin G) from rabbit serum by sequential
precipitation with caprylic acid and ammonium sulfate
One volume of rabbit serum is diluted with 4 volumes of acetate buffer (60 mM,
pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 ~1/ml
of
diluted sample) is added drop-wise under vigorous stirring. After 30 min the
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sample is centrifuged ( 13,000 x g, 30 min, 4°C), the pellet discarded
and the
supernatant collected. The pH of the supernatant is adjusted to 7.5 by the
addition
of 2 M Tris-base and filtered (0.2 ~tm).
The immunoglobulin in the supernatant is precipitated under vigorous stirring
by
the drop-wise addition of a 4 M ammonium sulfate solution to a final
concentration of 2 M. The precipitated immunoglobulins are collected by
centrifugation (8,000 x g, 15 min, 4°C).
The supernatant is discarded. The pellet is dissolved in 10 mM NaH2P04/NaOH,
pH 7.5, 30 mM NaCI and exhaustively dialyzed. The dialysate is centrifuged
(13,000 x g, 15 min, 4°C) and filtered (0.2 ~tm).
Biotinylation of polyclonal rabbit I~G
Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaHZPO4/NaOH, pH 7.5,
30 mM NaCI. Per ml IgG solution 50 ~.1 Biotin -N-hydroxysuccinimide (3.6 mg/ml
in DMSO) are added. After 30 min at room temperature, the sample is
chromatographed on Superdex 200 ( 10 mM NaHZPO4/NaOH, pH 7.5, 30 mM
NaCI). The fraction containing biotinylated IgG are collected. Monoclonal
antibodies are biotinylated according to the same procedure.
Digoxy enylation of polyclonal rabbit Ig_G
Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaHZPOø/NaOH, 30 mM
NaCI, pH 7.5. Per ml IgG solution 50 ~.1 digoxigenin-3-O-methylcarbonyl-E-
aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim,
Germany, Cat. No. 1 333 054) (3.8 mg/ml in DMSO) are added. After 30 min at
room temperature, the sample is chromatographed on Superdex~ 200 ( 10 mM
NaH2P04/NaOH, pH 7.5, 30 mM NaCI). The fractions containing digoxigenylated
IgG are collected. Monoclonal antibodies are labeled with digoxigenin
according to
the same procedure.
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Example 3
Western Blotting for the detection of SAHH in human colorectal cancer tissue
using polyclonal antibody as generated in Example 2.
Tissue lysates from tumor samples and healthy control samples are prepared as
described in Example 1, "Tissue preparation".
SDS-PAGE and Western-Blotting are carried out using reagents and equipment of
Invitrogen, Karlsruhe, Germany. For each tissue sample tested, 10 ~g of tissue
lysate
are diluted in reducing NuPAGE~ (Invitrogen) SDS sample buffer and heated for
ZO min at 95°C. Samples are run on 4-12% NuPAGE~ gels (Tris-Glycine)
in the
MES running buffer system. The gel-separated protein mixture is blotted onto
nitrocellulose membranes using the Invitrogen XCell IITM Blot Module
(Invitrogen)
and the NuPAGE° transfer buffer system. The membranes are washed 3
times in
PBS/0.05% Tween-20 and blocked with Roti~-Block blocking buffer (A151.1; Carl
Roth GmbH, Karlsruhe, Germany) for 2 h. The primary antibody, polyclonal
rabbit
anti-SAHH serum (generation described in Example 2), is diluted 1:10,000 in
Roti~-Bloclc blocking buffer and incubated with the membrane for 1 h. The
membranes are washed 6 times in PBS/0.05% Tween-20. The specifically bound
primary rabbit antibody is labeled with a POD-conjugated polyclonal sheep anti-
rabbit IgG antibody, diluted to 10 mU/ml in 0.5 x Roti~-Block blocking buffer.
After incubation for 1 h, the membranes are washed 6 times in PBS/0.05% Tween-
20. For detection of the bound POD-conjugated anti-rabbit antibody, the
membrane is incubated with the Lumi-LightPLUS Western Blotting Substrate
(Order-No. 2015196, Roche Diagnostics GmbH, Mannheim, Germany) and
exposed to an autoradiographic film.
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Results of a typical experiment are shown in Figure 2. The strong
overexpression of
SAHH in tumor tissue versus adjacent control tissue is found in 11 out of 14
subjects with colorectal cancer tested.
Example 4
ELISA for the measurement of SAHH in human stool specimen.
a) Processing of stool specimen
About 1 g of stool per sample is collected by the patients in a special
sampling
device from Sarstedt, Germany, Order-No. 80.623.022, frozen and stored at -20
°C.
For analysis, the stool samples are thawed, tenfold diluted with a phosphate
buffer,
pH 7.4 and thoroughly mixed in order to yield a suspension of the stool sample
in
the extraction buffer. After centrifugation for 15 min at 12,000 x g, the
upper
portion of the supernatant is saved for further analysis. An aliquot of this
processed
stool sample is used for quantification of SAHH by ELISA.
b) Measurement of SAHH from a processed stool sample
For detection of SAHH in human a processed stool sample, a sandwich ELISA is
developed. For capture and detection of the antigen, aliquots of the anti-SAHH
polyclonal antibody (see example 2) are conjugated with biotin and
digoxygenin,
respectively.
Streptavidin-coated 96-well microtiter plates are incubated with 100 ~tl
biotinylated
anti-SAHH polyclonal antibody for 60 min at 10 ~.g/ml in 10 mM phosphate,
pH 7.4, 1% BSA, 0.9% NaCI and 0.1% Tween-20. After incubation, plates are
washed three times with 0.9% NaCI , 0.1% Tween-20. Wells are then incubated
for
2 h with either a serial dilution of the recombinant protein (see Example 2)
as
standard antigen or with diluted stool samples from patients. After binding of
SAHH, plates are washed three times with 0.9% NaCI, 0.1% Tween-20. For
specific
detection of bound SAHH, wells are incubated with 100 ~l of digoxygenylated
anti-
SAHH polyclonal antibody for 60 min at 10 ~tg/ml in 10 mM phosphate, pH 7.4,
1% BSA, 0.9% NaCI and 0.1% Tween-20. Thereafter, plates are washed three times
to remove unbound antibody. In a next step, wells are incubated with 20 mU/ml
anti-digoxigenin-POD conjugates (Ruche Diagnostics GmbH, Mannheim,
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Germany, Catalog No. 1633716) for 60 min in 10 mM phosphate, pH 7.4, 1% BSA,
0.9% NaCI and 0.1% Tween-20. Plates are subsequently washed three times with
the same buffer. For detection of antigen-antibody complexes, wells are
incubated
with 100 ~1 ABTS solution (Ruche Diagnostics GmbH, Mannheim, Germany,
Catalog No. 11685767) and OD is measured after 30-60 min at 405 nm with an
ELISA reader.
Example 55
ROC analysis to assess clinical utility in terms of diagnostic accuracy
Accuracy is assessed by analyzing individual stool samples obtained from well-
characterized patient cohorts, i.e., 30 patients having undergone colonoscopy
and
found to be free of adenoma or CRC, 30 patients diagnosed and staged as Tl-3,
N0,
MO of CRC, and 30 patients diagnosed with progressed CRC, having at least
tumor
infiltration in at least one proximal lymph node or more severe forms of
metastasis,
respectively. SAHH is measured as described above in a stool sample obtained
from
each of these individuals. ROC-analysis is performed according to Zweig, M.
H.,
and Campbell, supra. Discriminatory power for differentiating patients in the
group
Tis-3, N0, MO from healthy individuals for the combination of SAHH with the
established marker hemoglobin (Hemoglobin ELISA; Catalogue No.: I~7816;
Immundiagnostik AG, Wiesenstr. 4, D 64625 Bensheim, Germany) is calculated by
regularized discriminant analysis (Friedman, J. H., Regularized Discriminant
Analysis, Journal of the American Statistical Association 84 (1989) 165-175).
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Tijssen, P., Practice and theory of enzyme immunoassays 11 ( 1990) the whole
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Turner, M.A., et al., Cell. Biochem. Biophys. 33 (2000) 101-125
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