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USE OF INORGANIC PYROPHOSPHATASE AS A MARKER FOR COLORECTAL CANCER
The present invention relates to the diagnosis of colorectal cancer. It
discloses the
use of IPYR (= inorganic pyrophosphatase) in the diagnosis of colorectal
cancer.
Furthermore, it especially relates to a method for diagnosis of colorectal
cancer
from a liquid sample, derived from an individual by measuring IPYR in said
sample. Measurement of IPYR can, e.g., be used in the early detection of
colorectal
cancer or in the surveillance of patients who undergo surgery.
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.
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),
edition, 1997 (Sobin, L.H., and Fleming, I.D., TNM 80 (1997) 1803-4).
What is especially important is that early diagnosis of CRC translates to a
much
better prognosis. Malignant tumors of the colorecturn 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 T;s, NO, MO or T1-3;
NO; MO,
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.
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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, NO, MO or T1-4; NO; MO are
present. Ts denotes carcinoma in situ.
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, NO,
MO or T1-3; NO; MO (=T;S-3; NO; MO).
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. 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).
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
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diagnostic sensitivity and/or specificity of a test is best assessed by its
receiver-
operating characteristics, which will be described in detail below.
The clinical utility of biochemical markers in colorectal cancer has recently
been
reviewed by the European Group on Tumor Markers (EGTM) (Duffy, M.J., et al
Europ. J. of Cancer 39 (2003) 718-727).
At present, primarily diagnostic blood tests based on the detection of
carcinoembryonic antigen (CEA), a tumor-associated glycoprotein, are available
to
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).
Whole blood, serum or plasma are the most widely used sources of sample in
clinical routine. The identification of an early CRC tumor marker that would
aid in
the reliable cancer detection or provide early prognostic information 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 in
vitro
assessment of CRC. 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.
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It was the task of the present invention to investigate whether a biochemical
marker
can be identified which may be used in assessing CRC.
Surprisingly, it has been found that use of the marker IPYR can at least
partially
overcome the problems known from the state of the art.
The present invention therefore relates to a method for assessing colorectal
cancer
in vitro by biochemical markers comprising measuring in a sample the
concentration of a) IPYR, and b) using the concentration determined in step
(a) in
the assessment of colorectal cancer.
Another preferred embodiment of the invention is a method for assessing
colorectal
cancer comprising the steps of a) contacting a liquid sample obtained from an
individual with a specific binding agent for IPYR under conditions appropriate
for
formation of a complex between said binding agent and IPYR, and b) correlating
the amount of complex formed in (a) to the assessment of colorectal cancer.
Yet another preferred embodiment of the invention relates to a method for
assessing colorectal cancer in vitro by biochemical markers, comprising
measuring
in a sample the concentration of IPYR and of one or more other marker of
colorectal cancer and using the concentrations determined in the assessment of
colorectal cancer.
The present invention also relates to the use of a marker panel comprising at
least
IPYR and CYFRA 21-1 in the assessment of CRC.
The present invention also relates to the use of a marker panel comprising at
least
IPYR and NSE in the assessment of CRC.
The present invention also relates to the use of a marker panel comprising at
least
IPYR and CEA in the assessment of CRC.
The present invention also relates to the use of a marker panel comprising at
least
IPYR and NNMT in the assessment of CRC.
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The present invention also relates to the use of a marker panel comprising at
least
IPYR and CA 19-9 in the assessment of CRC.
The present invention also relates to the use of a marker panel comprising at
least
IPYR and CA 72-4 in the assessment of CRC.
The present invention also provides a kit for performing the method according
to
the present invention comprising at least the reagents required to
specifically
measure IPYR and CYFRA 21-1, respectively, and optionally auxiliary reagents
for
performing the measurement.
The present invention also provides a kit for performing the method according
to
the present invention comprising at least the reagents required to
specifically
measure IPYR and NSE, respectively, and optionally auxiliary reagents for
performing the measurement.
In a further preferred embodiment the present invention relates to a method
for
assessing colorectal cancer in vitro comprising measuring in a sample the
concentration of a) IPYR, b) optionally one or more other marker of colorectal
cancer, and c) using the concentrations determined in step (a) and optionally
step
(b) in the assessment of colorectal cancer.
As used herein, each of the following terms has the meaning associated with it
in
this section.
The articles "a" and "an" are used herein to refer to one or to more than one
(i.e. to
at least one) of the grammatical object of the article. By way of example, "a
marker"
means one marker or more than one marker.
The term "marker" or "biochemical marker" as used herein refers to a molecules
to
be used as a target for analyzing patient test samples. Examples of such
molecular
targets are proteins or polypeptides themselves as well as antibodies present
in a
sample. Proteins or polypeptides used as a marker in the present invention are
contemplated to include any variants of said protein as well as fragments of
said
protein or said variant, in particular, immunologically detectable fragments.
One of
skill in the art would recognize that proteins which are released by cells or
present
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in the extracellular matrix which become damaged, e.g., during inflammation
could
become degraded or cleaved into such fragments. Certain markers are
synthesized
in an inactive form, which may be subsequently activated by proteolysis. As
the
skilled artisan will appreciate, proteins or fragments thereof may also be
present as
part of a complex. Such complex also may be used as a marker in the sense of
the
present invention. Variants of a marker polypeptide are encoded by the same
gene,
but differ in their PI or MW, or both (e.g., as a result of alternative mRNA
or pre-
mRNA processing, e.g. alternative splicing or limited proteolysis) and in
addition,
or in the alternative, may arise from differential post-translational
modification
(e.g., glycosylation, acylation, and/or phosphorylation).
The term "assessing colorectal cancer" is used to indicate that the method
according to the present invention will (alone or together with other markers
or
variables, e.g., the criteria set forth by the UICC (UICC (International Union
Against Cancer), Sobin, L.H., Wittekind, Ch. (eds), TNM Classification of
Malignant Tumours, fifth edition, 1997)) e.g., aid the physician to establish
or
confirm the absence or presence of CRC or aid the physician in the prognosis,
the
detection of recurrence (follow-up of patients after surgery) and/or the
monitoring
of treatment, especially of chemotherapy.
The term "sample" as used herein refers to a biological sample obtained for
the
purpose of evaluation in vitro. In the methods of the present invention, the
sample
or patient sample preferably may comprise any body fluid. Preferred test
samples
include blood, serum, plasma, urine, saliva, and synovial fluid. Preferred
samples
are whole blood, serum, plasma or synovial fluid, with plasma or serum being
most
preferred. As the skilled artisan will appreciate, any such assessment is made
in
vitro. The patient sample is discarded afterwards. The patient sample is
solely used
for the in vitro method of the invention and the material of the patient
sample is
not transferred back into the patient's body. Typically, the sample is a
liquid
sample, e.g., whole blood, serum, or plasma.
In a preferred embodiment the present invention relates to a method for
assessing
CRC in vitro by biochemical markers, comprising measuring in a sample the
concentration of IPYR and using the concentration determined in the assessment
of
CRC.
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The protein IPYR (inorganic pyrophosphatase, also known as pyrophosphate
phospho-hydrolase (PPase); HSPC124; MGC49850; PPA2; PPase; SID6 306) is
characterized by the sequences given SEQ ID NO:1, SEQ ID NO:2 or an isoform.
The protein inorganic pyrophosphatase is essential for the catalytic activity
of
PPase. PPases catalyze the hydrolysis of pyrophosphate to inorganic phosphate,
which is important for the phosphate metabolism of cells.
As obvious to the skilled artisan, the present invention shall not be
construed to be
limited to the full-length protein IPYR of SEQ ID NO: 1. Physiological or
artificial
fragments of IPYR, secondary modifications of IPYR, as well as allelic
variants of
IPYR are also encompassed by the present invention. Artificial fragments
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.
Preferably, full-length IPYR or a physiological variant of this marker is
detected in a
method according to the present invention.
The assessment method according to the present invention is based on a liquid
sample which is derived from an individual. Unlike to methods known from the
art
IPYR is specifically measured from this liquid sample by use of a specific
binding
agent.
A specific binding agent is, e.g., a receptor for IPYR, a lectin binding to
IPYR or an
antibody to IPYR. 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 10$ 1/mol or even more preferred of 109 1/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 the binding
agent
specific for IPYR. Preferably, the level of binding to a biomolecule other
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
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binding agent will fulfill both the above minimum criteria for affinity as
well as for
specificity.
A specific binding agent preferably is an antibody binding to IPYR. 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 IPYR in a
method according to the present invention is yet another preferred embodiment.
As the skilled artisan will appreciate now, that IPYR 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 IPYR
for
immunization. Alternatively, DNA Immunization also known as DNA vaccination
may be used.
For measurement the liquid sample obtained from an individual is incubated
with
the specific binding agent for IPYR under conditions appropriate for formation
of a
binding agent IPYR-complex. Such conditions need not be specified, since the
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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 the specific binding agent IPYR-complex all described in detail in relevant
textbooks (cf., e.g., Tijssen P., supra, or Diamandis, et al., eds. (1996)
Immunoassay,
Academic Press, Boston).
Preferably IPYR is detected in a sandwich type assay format. In such assay a
first
specific binding agent is used to capture IPYR 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 IPYR can be measured
from a liquid sample obtained from an individual sample. No tissue and no
biopsy
sample is required to apply the marker IPYR in the assessment of CRC.
In a preferred embodiment the method according to the present invention is
practiced with serum as liquid sample material. In a further preferred
embodiment
the method according to the present invention is practiced with plasma as
liquid
sample material. In a further preferred embodiment the method according to the
present invention is practiced with whole blood as liquid sample material.
Furthermore stool can be prepared in various ways known to the skilled artisan
to
result in a liquid sample as well. Such sample liquid derived from stool also
represents a preferred embodiment according to the present invention.
The inventors of the present invention have surprisingly been able to detect
protein
IPYR in a bodily fluid sample. Even more surprising they have been able to
demonstrate that the presence of IPYR in such liquid sample obtained from an
individual can be correlated to the assessment of colorectal cancer.
Preferably, an
antibody to IPYR is used in a qualitative (IPYR present or absent) or
quantitative
(IPYR amount is determined) immunoassay.
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Measuring the level of protein IPYR has proven very advantageous in the field
of
CRC. Therefore, in a further preferred embodiment, the present invention
relates
to use of protein IPYR as a marker molecule in the assessment of colorectal
cancer
from a liquid sample obtained from an individual.
The ideal scenario for diagnosis would be a situation wherein a single event
or
process would cause the respective disease as, e.g., in infectious diseases.
In all other
cases correct diagnosis can be very difficult, especially when the etiology of
the
disease is not fully understood as is the case of CRC. As the skilled artisan
will
appreciate, no biochemical marker, for example in the field of CRC, is
diagnostic
with 100% specificity and at the same time 100% sensitivity for a given
disease.
Rather, biochemical markers are used to assess with a certain likelihood or
predictive value the presence or absence of a disease. Therefore, in routine
clinical
diagnosis various clinical symptoms and biological markers are generally
considered together in the diagnosis, treatment, and management of the
underlying
disease.
Biochemical markers can either be determined individually or, in a preferred
embodiment of the invention, they can be measured simultaneously using a chip-
or a bead-based array technology. The concentrations of the biomarkers are
then
interpreted independently using an individual cut-off for each marker or they
are
combined for interpretation.
In a further preferred embodiment of the invention the assessment of
colorectal
cancer according to the present invention is performed in a method comprising
measuring in a sample the concentration of a) IPYR, b) optionally one or more
other marker of colorectal cancer, and c) using the concentration determined
in
step (a) and optionally step (b) in the assessment of colorectal cancer.
Preferably the method for assessment of CRC is performed by measuring the
concentration of IPYR and of one or more other marker and by using the
concentration of IPYR and of the one or more other marker in the assessment of
CRC.
The present invention is also directed to a method for assessing CRC in vitro
by
biochemical markers, comprising measuring in a sample the concentration of
IPYR
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and of one or more other marker of CRC and using the concentrations determined
in the assessment of CRC.
According to the data shown in the Example section the marker IPYR in the
univariate analysis has (at a specificity of about 90%) a sensitivity for CRC
of
54.7%. In the assessment of CRC the marker IPYR will be of advantage in one or
more of the following aspects: screening; diagnostic aid; prognosis;
monitoring of
chemotherapy, and follow-up.
Screenin~=
~
CRC is the second most common malignancy of both males and females in
developed countries. Because of its high prevalence, its long asymptomatic
phase
and the presence of premalignant lesions, CRC meets many of the criteria for
screening. Clearly, a serum tumour marker which has acceptable sensitivity and
specificity would be more suitable for screening than either FOB testing or
endoscopy.
As the data given in the Examples section demonstrate IPYR alone will not
suffice
to allow for a general screening e.g. of the at risk population for CRC. Most
likely
no single biochemical marker in the circulation will ever meet the sensitivity
and
specificity criteria required for screening purposes. Rather it has to be
expected that
a marker panel will have to be used in CRC screening. The data established in
the
present invention indicate that the marker IPYR will form an integral part of
a
marker panel appropriate for screening purposes. The present invention
therefore
relates to the use of IPYR as one marker of a CRC marker panel for CRC
screening
purposes. The present data further indicate that certain combinations of
markers
will be advantageous in the screening for CRC. Therefore the present invention
also
relates to the use of a marker panel comprising IPYR and CYFRA 21-1, or of a
marker panel comprising IPYR and NSE, or of a marker panel comprising IPYR
and CYFRA 21-1 and NSE for the purpose of screening for CRC.
Diagnostic aid:
Preoperative CEA values are of limited diagnostic value. Nonetheless the
European
Committee on Tumor Markers (ECTM) recommends that CFA should be
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measured before surgery in order to establish a baseline value and for
assessing the
prognosis. Since IPYR as a single marker according to the data of the present
invention might be at least as good a single marker as CEA or even superior it
has to
be expected that IPYR will be used as a diagnostic aid, especially by
establishing a
baseline value before surgery.
The present invention thus also relates to the use of IPYR for establishing a
baseline
value before surgery for CRC.
Pro ng osis:
The gold standard for determining prognosis in patients with CRC is the extend
of
disease as defined by the Dukes', TNM or other staging systems, If a marker
such as
CEA is to be used for predicting outcome, it must: provide stronger prognostic
information than that offered by existing staging systems, provide information
independent of the existing systems or provide prognostic data within specific
subgroups defined by existing criteria, e.g. in Dukes' B or node-negative,
patients.
Recently, an American Joint Committee on Cancer (AJCC) Consensus Conference
suggested that CEA should be added to the TNM staging system for colorectal
cancer. The CEA level should be designated as follows: CX, CEA cannot be
assessed;
CO, CEA not elevated (<5 g/1) or CEA1, CEA elevated (> 5 g/1) (Compton, C.,
et
al., Cancer 88 (2000) 1739-1757).
As IPYR alone significantly contributes to the differentiation of CRC patients
from
healthy controls or from healthy controls plus non-malignant colon diseases,
it has
to be expected that it will aid in assessing the prognosis of patients
suffering from
CRC. The level of preoperative IPYR will most likely be combined with one or
more other marker for CRC and/or the TNM staging system, as recommended for
CEA by the AJCC. In a preferred embodiment IPYR is used in the prognosis of
patients with CRC.
Monitoring of Chemotherap~::
A number of reports have described the use of CEA in monitoring the treatment
of
patients with advanced CRC (for review, see Refs. Duffy, M.J., Clin. Hem. 47
(2001)
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625-630; Fletcher, R.H., Ann. Int. Med. 104 (1986) 66-73; Anonymous, J. Clin.
Oncol. 14 (1996) 2843-2877). Most of these were retrospective, non-randomized
and contained small numbers of patients. These studies suggested: a) that
patients
with a decrease in CEA levels while receiving chemotherapy generally had a
better
outcome than those patients whose CEA levels failed to decrease and (b) for
almost
all patients, increases in CEA levels were associated with disease
progression.
Due to the data shown in the example section, it has to be expected that IPYR
will
be at least as good a marker for monitoring of chemotherapy as CEA. The
present
invention therefore also relates to the use of IPYR in the monitoring of CRC
patients under chemotherapy.
Follow-uy=
~
Approximately 50 % of patients who undergo surgical resection aimed at cure,
later
develop recurrent of metastatic disease (Berman, J.M., et al., Lancet 355
(2000)
395-399). Most of these relapses occur within the first 2-3 years of diagnosis
and are
usually confined to the liver, lungs or locoregional areas. Since
recurrent/metastatic
disease is invariably fatal, considerable research has focused on its
identification at
an early and thus potentially treatable stage. Consequently, many of these
patients
undergo a postoperative surveillance program which frequently includes regular
monitoring with CEA.
Serial monitoring with CEA has been shown to detect recurrent/metastatic
disease
with a sensitivity of approximately of 80 %, specificity of approximately 70 %
and
provides an average lead-time of 5 months (for review, see Duffy, M.J., et
al., supra
and Fletcher, R.H., supra). Furthermore, CEA was the most frequent indicator
of
recurrence in asymptomatic patients (Pietra, N., et al., Dis. Colon Rectum 41
(1998) 1127-1133 and Graham, R.A., et al., Ann. Surg. 228 (1998) 59-63) and
was
more cost-effective than radiology for the detection of potentially curable
recurrent
disease. As regards sites of recurrence/metastasis, CEA was most sensitive
(almost
100%) for the detection of liver metastasis. On the other hand, CEA was less
reliable
for diagnosing locoregional recurrences, the sensitivity being only
approximately 60
% (Moertel, C.G., et al., JAMA 270 (1993)943-7)
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As a compromise between patient convenience, costs and efficiency of disease
detection, the EGTM Panel like the IPYRO Panel (Anonymous, J. Clin. Oncol. 14
(1996) 2843-2877) suggests that CEA testing be carried out every 2-3 months
for at
least 3 years after the initial diagnosis. After 3 years, testing could be
carried out less
frequently, e.g. every 6 months. No evidence exists, however, to support this
frequency of testing.
As the above discussion of the state of the art shows, that the follow-up of
patients
with CRC after surgery is one of the most important fields of use for an
appropriate
biochemical marker. Due to the high sensitivity of IPYR in the CRC patients
investigated it is expected that IPYR alone or in combination with one or more
other marker will be of great help in the follow-up of CRC patients,
especially in
CRC patients after surgery. The use of a marker panel comprising IPYR and one
or
more other marker of CRC in the follow-up of CRC patients represents a further
preferred embodiment of the present invention.
The present invention discloses and therefore in a preferred embodiment
relates to
the use of IPYR in the diagnostic field of CRC or in the assessment of CRC,
respectively.
In yet a further preferred embodiment the present invention relates to the use
of
IPYR as a marker molecule for colorectal cancer in combination with one or
more
marker molecules for colorectal cancer in the assessment of colorectal cancer
from
a liquid sample obtained from an individual. In this regard, the expression
"one or
more" denotes 1 to 20, preferably 1 to 10, preferably 1 to 5, more preferred 3
or 4.
IPYR and the one or more other marker form a CRC marker panel.
Thus, a preferred embodiment of the present invention is the use of IPYR as a
marker molecule for colorectal cancer in combination with one or more marker
molecules for colorectal cancer in the assessment of colorectal cancer from a
liquid
sample obtained from an individual. Preferred selected other CRC markers with
which the measurement of IPYR may be combined are NSE, CYFRA 21-1, NMMT,
CA 19-9, CA 72-4, and/or CEA. Yet further preferred the marker panel used in
the
assessment of CRC comprises IPYR and at least one other marker molecule
selected
from the group consisting of NSE, CYFRA 21-1 and NMMT.
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The markers which preferably are combined with IPYR or which form part of the
CRC marker panel comprising IPYR, respectively, are discussed in more detail
below.
NSE (neuron-specific enolase)
The glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11,
molecular weight approx. 80 kD) occurs in a variety of dimeric isoforms
comprising three immunologically different subunits termed a, (3, and y. The a-
subunit of enolase occurs in numerous types of tissue in mammals, whereas the
(3-
subunitis found mainly in the heart and in striated musculature. The enolase
isoforms ay and yy, which are referred to as neuron-specific enolase (NSE) or -
y-
enolase, are primarily detectable in high concentrations in neurons and neuro-
endocrine cells as well as in tumors originating from them (Lamerz, R., NSE
(Neuronen-spezifische Enolase), y-Enolase, In: Thomas L (ed) Clinical
Laboratory
Diagnosis, TH-Books, Frankfurt, 1St English Edition 1998: 979-981, 5. deutsche
Auflage 1998:1000-1003).
NSE is described as the marker of first choice in the monitoring of small cell
bronchial carcinoma (Lamerz, R., supra), whereas CYFRA 21-1 is superior to NSE
for non-small cell bronchial carcinoma (Ebert, W., et al., Eur. J. Clin. Chem.
Clin.
Biochem 32 (1994) 189-199).
Elevated NSE concentrations are found in 60-81 % of cases of small cell
bronchial
carcinoma.
For NSE there is no correlation to the site of metastasis or to cerebal
metastasis, but
there is good correlation to the clinical stage, i.e. the extent of the
disease.
In response to chemotherapy there is a temporary rise in the NSE leve124-72
hours
after the first therapy cycle as a result of cytolysis of the tumor cells.
This is followed
within a week or by the end of the first therapy cycle by a rapid fall in the
serum
values (which were elevated prior to therapy). By contrast, non-responders to
therapy display levels which are constantly elevated or fail to fall into the
reference
range. During remission, 80-96 % of the patients have normal values. Rising
NSE
values are found in cases of relapse. The rise occurs in some cases with a
latent
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period of 1-4 months, is often exponential (with a doubling time of 10-94
days) and
correlates with the survival period. NSE is useful as a single prognostic
factor and
activity marker during the monitoring of therapy and the course of the disease
in
small cell bronchial carcinoma: diagnostic sensitivity 93 %, positive
predictive value
92% (Lamerz, R., supra).
In neuroblastoma NSE serum values above 30 ng/ml are found in 62 % of the
affected children. The medians rise in accordance with the stage of the
disease.
There is a significant correlation between the magnitude or frequency of
pathological NSE values and the stage of disease; there is an inverse
correlation with
illness-free survival.
68-73 % of the patients with seminoma have a clinically significant NSE
elevation
(Lamerz, R., supra). There is a utilizable correlation with the clinical
course of the
disease.
NSE has also been measured in other tumors: Non-pulmonary malignant diseases
show values above 25 ng/ml in 22 % of the cases (carcinomas in all stages).
Brain
tumors such as glioma, miningioma, neurofibroma, and neurinoma are only
occasionally accompanied by elevated serum NSE values. In primary brain tumors
or brain metastasis and in malignant melanoma and phaeochromocytoma, elevated
NSE-values can occur in the CSF (cerebrospinal fluid). Increased NSE
concentrations have been reported for 14 % of organ-restricted and 46 % of
metastasizing renal carcinomas, with a correlation to the grade as an
independent
prognosis factor.
In benign disease elevated serum NSE concentrations (> 12 ng/ml) have been
found
in patients with benign pulmonary diseases and cerebral diseases. Elevated
values,
mainly in the liquor, have been found in cerebrovascular meningitis,
disseminated
encephalitis, spinocerebellar degeneration, cerebral ischemia, cerebral
infarction,
intracerebral hematoma, subarachnoid hemorrhage, head injuries, inflammatory
brain diseases, organic epilepsy, schizophrenia, and Jakob-Creutzfeld disease
(Lamerz, R., supra).
NSE has been measured on an Elecsys analyzer using Roche product number
12133113 according to the manufacturers instructions.
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CA 19-9 Carbohydrate A.nti eg n 19-9
The CA 19-9 values measured are defined by the use of the monoclonal antibody
1116-NS-19-9. The 1116-NS-19-9-reactive determinants on a glycolipid having a
molecular weight of approx. 10,000 daltons are measured. This mucin
corresponds
to a hapten of Lewis-a blood group determinants and is a component of a number
of mucous membrane cells (Koprowski, H., et al., Somatic Cell Genet 5 (1979)
957-
971).
3-7 % of the population have the Lewis a-negative/b-negative blood group
configuration and are unable to express the mucin with the reactive
determinant
CA 19-9. This must be taken into account when interpreting the findings.
Mucin occurs in fetal gastric, intestinal and pancreatic epithelia. Low
concentrations can also be found in adult tissue in the liver, lungs, and
pancreas
(Stieber, P., and Fateh-Moghadam, A., Boeringer Mannheim, Cat. No. 1536869
(engl), 1320947 (dtsch), ISBN 3-926725-07-9 dtsch/engl. Juergen Hartmann
Verlag
Marloffstein-Rathsberg (1993); Herlyn, M., et al., J. Clin. Immunol 2 (1982)
135-
140).
CA 19-9 assay values can assist in the differential diagnosis and monitoring
of
patients with pancreatic carcinoma (sensitivity 70-87 %) (Ritts, R.E., Jr., et
al., Int.
J. Cancer 33 (1984) 339-345). There is no correlation between tumor mass and
the
CA 19-9 assay values. However, patients with CA 19-9 serum levels above 10,000
U/mL almost always have distal metastasis.
The determination of CA 19-9 cannot be used for the early detection of
pancreatic
carcinoma (Steinberg, W.M., et al., Gastroenterology 90 (1986) 343-349).
In hepatobiliary carcinoma the CA 19-9 values provide a sensitivity of 50-75
%. The
concomitant determination of CA 72-4 and CEA is recommended in case of gastric
carcinoma. In colorectal carcinoma, determination of CEA alone is adequate;
only
in rare CEA-negative cases the determination of CA 19-9 can be useful.
As the mucin is excreted exclusively via the liver, even slight cholestasis
can lead to
clearly elevated CA 19-9 serum levels in some cases. Elevated CA 19-9 values
are
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also found with a number of benign and inflammatory diseases of the
gastrointestinal tract and the liver, as well as in cystic fibrosis.
CA 19-9 has been measured on an Elecsys analyzer using Roche product number
11776193 according to the manufacturers instructions.
CEA Carcinoembrõyonic antigen
CEA is a monomeric glycoprotein (molecular weight approx. 180.000 dalton) with
a variable carbohydrate component of approx. 45-60 % (Gold, P., and Freedman,
S.O., J. Exp. Med. 121 (1965) 439-462).
CEA, like AFP, belongs to the group of carcinofetal antigens that are produced
during the embryonic and fetal period. The CEA gene family consists of about
17
active genes in two subgroups. The first group contains CEA and the Non-
specific
Cross-reacting Antigens (NCA); the second group contains the Pregnancy-
Specific
Glycoproteins (PSG).
CEA is mainly found in the fetal gastrointestinal tract and in fetal serum. It
also
occurs in slight quantities in intestinal, pancreatic, and hepatic tissue of
healthy
adults. The formation of CEA is repressed after birth, and accordingly serum
CEA
values are hardly measurable in healthy adults.
High CEA concentrations are frequently found in cases of colorectal
adenocarcinoma (Stieber, P., and Fateh-Moghadam, A., supra). Slight to
moderate
CEA elevations (rarely > 10 ng/mL) occur in 20-50 % of benign diseases of the
intestine, the pancreas, the liver, and the lungs (e.g. liver cirrhosis,
chronic hepatitis,
pancreatitis, ulcerative colitis, Crohn's Disease, emphysema) (Stieber, P.,
and
Fateh-Moghadam, A., supra). Smokers also have elevated CEA values.
The main indication for CEA determinations is the follow-up and therapy
management of colorectal carcinoma.
CEA determinations are not recommended for cancer-screening in the general
population. CEA concentrations within the normal range do not exclude the
possible presence of a malignant disease.
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The antibodies in assay manufactured by Roche Diagnostics react with CEA and
(as with almost all CEA methods) with the meconium antigen (NCA2). Cross-
reactivity with NCA1 is 0.7 % (Hammarstrom, S., et al., Cancer Research 49
(1989)
4852-4858 and Bormer, O.P., Tumor Biol. 12 (1991) 9-15).
CEA has been measured on an Elecsys analyzer using Roche product number
11731629 according to the manufacturers instructions.
CYFRA 21-1
An assay for "CYFRA 21-1" specifically measures a soluble fragment of
cytokeratin
19 as present in the circulation. The measurement of CYFRA 21-1 is typically
based
upon two monoclonal antibodies (Bodenmueller, H., et al., Int. J. Biol.
Markers 9
(1994) 75-81). In the CYFRA 21-1 assay from Roche Diagnostics, Germany, the
two
specific monoclonal antibodies (KS 19.1 and BM 19.21) are used and a soluble
fragment of cytokeratin 19 having a molecular weight of approx. 30,000 daltons
is
measured.
Cytokeratins are structural proteins forming the subunits of epithelial
intermediary
filaments. Twenty different cytokeratin polypeptides have so far been
identified.
Due to their specific distribution patterns they are eminently suitable for
use as
differentiation markers in tumor pathology. Intact cytokeratin polypeptides
are
poorly soluble, but soluble fragments can be detected in serum (Bodenmueller,
H.,
et al., supra).
CYFRA 21-1 is a well-established marker for Non-Small-Cell Lung Carcinoma
(NSCLC). The main indication for CYFRA 21-1 is monitoring the course of non-
small cell lung cancer (NSCLC) (Sturgeon, C., Clinical Chemistry 48 (2002)
1151-
1159).
High CYFRA 21-1 serum levels indicate an advanced tumor stage and a poor
prognosis in patients with non-small-cell lung cancer (van der Gaast A. et
al., Br. J.
Cancer 69 (1994) 525-528). A normal or only slightly elevated value does not
rule
out the presence of a tumor.
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Successful therapy is documented by a rapid fall in the CYFRA 21-1 serum level
into the normal range. A constant CYFRA 21-1 value or a slight or only slow
decrease in the CYFRA 21-1 value indicates incomplete removal of a tumor or
the
presence of multiple tumors with corresponding therapeutic and prognostic
consequences. Progression of the disease is often shown earlier by increasing
CYFRA 21-1 values than by clinical symptomatology and imaging procedures.
It is accepted that in the primary diagnosis of pulmonary carcinoma should be
made on the basis of clinical symptomatology, imaging or endoscopic procedures
and intraoperative findings. An unclear circular focus in the lung together
with
CYFRA. 21-1 values > 30 ng/mL indicates with high probability the existence of
primary bronchial carcinoma.
CYFRA. 21-1 is also suitable for course-monitoring in myoinvasive cancer of
the
bladder. Good specificity is shown by CYFRA 21-1 relative to benign lung
diseases
(pneumonia, sarcoidosis, tuberculosis, chronic bronchitis, bronchial asthma,
emphysema).
Slightly elevated values (up to 10 ng/mL) are rarely found in marked benign
liver
diseases and renal failure. There is no correlation with sex, age or smoking.
The
values for CYFRA 21-1 are also unaffected by pregnancy.
Recently it has been found that CYFRA 21-1 also is of use in detecting disease
relapse and assessing treatment efficacy in the field of breast cancer
(Nakata, B., et
al., British J. of Cancer (2004) 1-6).
CYFRA 21-1 has been measured on an Elecsys analyzer using Roche product
number 11820966 according to the manufacturers instructions.
As mentioned further above CYFRA 21-1 is an established marker in the field of
NSCLC. When developing and establishing CYFRA 21-1 for NSCLC, non-
malignant disease controls derived from patients with certain lung non-
malignant
diseases have been used. This has been considered important to differentiate
benign
from malign lung diseases (H. Bodenmuller, et al., supra).,
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Since only recently it is possible to detect the marker CYFRA 21-1 in a
significant
percentage of samples derived from patients with CRC. In addition, the
presence of
CYFRA 21-1 in such liquid sample obtained from an individual can be used in
the
assessment of colorectal cancer. Particularly in combination with other
markers
CYFRA 21-1 is considered to be a very useful marker in the field of CRC.
NMMT
The protein nicotinamide N-methyltransferase (NNMT; Swiss-PROT: P40261) has
an apparent molecular weight of 29.6 kDa and an isoelectric point of 5.56.
NNMT catalyzes the N-methylation of nicotinamide and other pyridines. This
activity is important for biotransformation of many drugs and xenobiotic
compounds. The protein has been reported to be predominantly expressed in
liver
and is located in the cytoplasm. NNMT has been cloned from cDNA from human
liver and contained a 792-nucleotide open reading frame that encoded a 264-
amino
acid protein with a calculated molecular mass of 29.6 kDa (Aksoy, S., et al.,
J. Biol.
Chem. 269 (1994) 14835-14840). Little is known in the literature about a
potential
role of the enzyme in human cancer. In one paper, increased hepatic NNMT
enzymatic activity was reported as a marker for cancer cachexia in mice
(Okamura,
A., et al., Jpn. J. Cancer Res. 89 (1998) 649-656). In a recent report, down-
regulation of the NNMT gene in response to radiation in radiation sensitive
cell
lines was demonstrated (Kassem, H., et al., Int. J. Cancer 101 (2002) 454-
460).
It has recently been found (WO 2004/057336) that NMMT will be of interest in
the
assessment of CRC. The immunoassay described in WO 2004/057336 has been used
to measure the samples (CRC, healthy controls and non-malignant colon
diseases)
of the present study.
As the skilled artisan will appreciate there are many ways to use the
measurements
of two or more markers in order to improve the diagnostic question under
investigation. In a quite simple, but nonetheless often effective approach, a
positive
result is assumed if a sample is positive for at least one of the markers
investigated.
This may e.g. the case when diagnosing an infectious disease, like AIDS.
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Frequently, however, the combination of markers is evaluated. Preferably the
values
measured for markers of a marker panel, e.g. for IPYR, CYFRA 21-1 and NSE, are
mathematically combined and the combined value is correlated to the underlying
diagnostic question. Marker values may be combined by any appropriate state of
the art mathematical method. Well-known mathematical methods for correlating a
marker combination to a disease employ methods like, discriminant analysis
(DA)
(i.e. linear-, quadratic-, regularized-DA), Kernel Methods (i.e. SVM),
Nonparametric Methods (i.e. k-Nearest-Neighbor Classifiers), PLS (Partial
Least
Squares), Tree-Based Methods (i.e. Logic Regression, CART, Random Forest
Methods, Boosting/Bagging Methods), Generalized Linear Models (i.e. Logistic
Regression), Principal Components based Methods (i.e. SIMCA), Generalized
Additive Models, Fuzzy Logic based Methods, Neural Networks and Genetic
Algorithms based Methods. The skilled artisan will have no problem in
selecting an
appropriate method to evaluate a marker combination of the present invention.
Preferably the method used in correlating the marker combination of the
invention
e.g. to the absence or presence of CRC is selected from DA (i.e. Linear-,
Quadratic-,
Regularized Discriminant Analysis), Kernel Methods (i.e. SVM), Nonparametric
Methods (i.e. k-Nearest-Neighbor Classifiers), PLS (Partial Least Squares),
Tree-
Based Methods (i.e. Logic Regression, CART, Random Forest Methods, Boosting
Methods), or Generalized Linear Models (i.e. Logistic Regression). Details
relating
to these statistical methods are found in the following references: Ruczinski,
I., et al,
J. of Computational and Graphical Statistics, 12 (2003) 475-511; Friedman,
J.H. , J.
of the American Statistical Association 84 (1989) 165-175; Hastie, Trevor,
Tibshirani, Robert, Friedman, Jerome, The Elements of Statistical Learning,
Springer Series in Statistics, 2001; Breiman, L., Friedman, J.H., Olshen,
R.A., Stone,
C.J. (1984) Classification and regression trees, California: Wadsworth;
Breiman, L.,
Random Forests, Machine Learning, 45 (2001) 5-32; Pepe, M.S., The Statistical
Evaluation of Medical Tests for Classification and Prediction, Oxford
Statistical
Science Series, 28 (2003); and Duda, R.O., Hart, P.E., Stork, D.G., Pattern
Classification, Wiley Interscience, 2nd Edition (2001).
It is a preferred embodiment of the invention to use an optimized multivariate
cut-
off for the underlying combination of biological markers and to discriminate
state
A from state B, e.g. diseased from healthy. In this type of analysis the
markers are
no longer independent but form a marker panel. It could be established that
combining the measurements of IPYR, NSE and CYFRA 21-1, does particularly
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improve the diagnostic accuracy for CRC as compared to either healthy controls
or,
as also assessed, as compared to healthy controls plus non-malignant disease
controls. Especially the later finding is of great importance, because a
patient with a
non-malignant disease may require quite a different treatment as a patient
with
CRC.
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/ 1 -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).
Combining measurements of IPYR with other recently discovered markers, like
CYFRA 21-1 or NMMT or with known markers like CEA and NSE, or with other
markers of CRC yet to be discovered, leads and will lead, respectively, to
further
improvements in assessment of CRC.
The following examples, references, and sequence listing 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.
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
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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
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 IPYR 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.
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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 MgC12, 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
4,500 x g.
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.
Sample preparation for LC-ESI-MSMS-analysis
The protein concentration of the soluble protein fraction is determined using
Bio-
Rad protein assay (Cat.No. 500-0006; Bio-Rad Laboratories GmbH, Munchen,
Germany) following the instructions of the supplier's manual. To a volume
corresponding to 200 g of protein 4 ml reduction buffer (9 M urea, 2 mM DTT,
100 mM KH2PO4, pH 8.2 NaOH) is added and incubated for 1 h. The solution is
concentrated to 250 l in an Amicon Ultra 10 kD device (Millipore GmbH,
Schwalbach, Germany). For alkylation the 250 l are transferred into 1 ml
alkylation buffer (9 M urea, 4 mM iodoacetamide, 100 mM KH2PO4, pH 8.2
NaOH), incubated for 6 h and subsequently concentrated in an Amicon Ultra
10 kD device to 250 l. For washing 1 ml 9 M urea is added and again
concentrated
in an Amicon Ultra 10 kD device to 250 1. Washing is repeated three-times.
For protease digestion the concentrated solution is diluted to 2.5 M urea and
incubated with 4 g trypsin (Proteomics grade, Roche Diagnostics GmbH,
Mannheim, Germany) over night. The digestion is stopped by adding 1 ml 1%
formic acid and analyzed.
LC-ESI-MSMS-analysis
The tryptic digest (500 l) is separated on a two-dimensional Nano-HPLC-System
(Ultimate, Famos, Switchos; LC Packings, Idstein, Germany) consisting of a SCX
and a RP Pepmep C18 column (LC Packings, Idstein, Germany). The 11 SCX
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fractions (step elution with 0, 5, 10, 25, 50, 100, 200, 300, 400, 500, 1,500
mM
NH4Ac) where successively further separated on the RP column with a 90 min
gradient (5-95% acetonitrile) and online analyzed using data dependent scans
with
an ESI-MS ion trap (LCQ deca XP; Thermo Electron, Massachusetts, USA; see
Table 2 for parameters). For each sample three runs are performed. The raw
data
are processed with Bioworks 3.1 software (Thermo Electron, Massachusetts, USA)
using the parameters listed in Table 2. The resulting lists of identified
peptides and
proteins from replicate runs where combined.
The protein IPYR is identified with the sequences given in Table 1.
Detection of IPYR as a potential marker for colorectal cancer
For each patient the identified proteins and the number of corresponding
peptides
from the tumor sample are compared to the accordant results from adjacent
normal tissue and from stripped normal mucosa tissue. By this means, protein
IPYR is found to be specifically expressed or strongly overexpressed in tumor
tissue
and not or less 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.
The protein IPYR is strongly over-represented in tumor tissue from patients
suffering from colorectal cancer. The peptide sequences of the protein IPYR
listed
in Table 1 has been identified with Bioworks 3.1 form LCQ-MS2-data in tumor
tissue:
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Table 1
i DVFHVVVGSTTLSNAKMEIATK
ii GISCM*NTTVSK
iii VIAINVDDPDAANCNDINDVK
iv YVANLFPYKGYIWNYGAIPQTWK
v AIVDALPPPCESACTVPTDVDKWFHHQK
vi GISCMNTTLSESPFK
vii GQYISPFHDIPIYADKDVFHMVVEVPR
viii GYIWNYGAIPQTWEDPGHNDK
ix HTGCCGDNDPIDVCEIGSK
x LKPGYLEATVDWFR
xi VIAINVDDPDAANYNDINDVK
xii VIAINVDDPDAANYNDINDVKR
xiii VLGILAMIDEGETDWK
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Table 2: MSMS-data acquisition and Bioworks 3.1 search parameters
MSMS-data MS exclusion 350-2,000 Da for precursor ions
acquisition
Repeat count 2
Repeat duration 0.25 min
Exclusion list size 25
Exclusion duration 5 min
Exclusion mass width low 0.5 Da, high 1.5 Da
Bioworks Number of ions 35
Minimal ion intensity 100,000 counts
Precursor mass tolerance 1.2 Da
Fragment mass tolerance 1.4 Da
Xcorr > 2; 2.5; 3(z = 1; 2; 3)
dCn > 0.1
Sp > 500
Databases Swissprot;
Humangp (assembled by Roche
Bioinformatics)
Example 2
Generation of antibodies to the colorectal cancer marker protein IPYR
Polyclonal antibody to the colorectal cancer marker protein IPYR is generated
for
further use of the antibody in the measurement of serum and plasma and blood
levels of IPYR by immunodetection assays, e.g. Western Blotting and ELISA.
Recombinant protein expression in E. coli
In order to generate antibodies to IPYR, 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.
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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 IPYR 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.
Purification of His-IPYR fusion protein is done following standard procedures
on a
Ni-chelate column. Briefly, 11 of bacteria culture containing the expression
vector
for the His-IPYR 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 .
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 IPYR
a) Immunization of mice
12 week old A/J mice are initially immunized intraperitoneally with 100 g
IPYR.
This is followed after 6 weeks by two further intraperitoneal immunizations at
monthly intervals. In this process each mouse is administered 100 g IPYR
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 IPYR in PBS buffer for each.
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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*108 spleen cells of the immunized mouse are mixed with
2x107
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 4,000, 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
10 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.
After ca. 10 days the primary cultures are tested for specific antibody. IPYR-
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
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
Bruck,
C., et al., Methods Enzymol. 121 (1986) 587-695).
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Generation of polyclonal antibodies
a) Immunization
For immunization, a fresh emulsion of the protein solution (100 g/ml protein
IPYR) 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-IPYR 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 l/ml
of
diluted sample) is added drop-wise under vigorous stirring. After 30 min the
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 m).
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 NaHzPO4/NaOH,
pH 7.5, 30 mM NaCl and exhaustively dialyzed. The dialysate is centrifuged
(13,000 x g, 15 min, 4 C) and filtered (0.2 m).
Biotinylation of polyclonal rabbit IgG
Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH2PO4/NaOH, pH 7.5,
30 mM NaCI. Per ml IgG solution 50 l 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 NaH2PO4/NaOH, pH 7.5, 30 mM
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NaC1). The fraction containing biotinylated IgG are collected. Monoclonal
antibodies are biotinylated according to the same procedure.
DigoxXgenylation of polyclonal rabbit IgG
Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH2PO4/NaOH, 30 mM
NaCI, pH 7.5. Per ml IgG solution 50 l 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
NaHZPO4/NaOH, pH 7.5, 30 mM NaCl). The fractions containing digoxigenylated
IgG are collected. Monoclonal antibodies are labeled with digoxigenin
according to
the same procedure.
Example 3
Western Blotting for the detection of IPYR 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
10 min at 95 C. Samples are run on 4-12% NuPAGEO 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-IPYR serum (generation described in Example 2), is diluted 1:10,000 in
Roti -
Block 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 an 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
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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.
Example 4
ELISA for the measurement of IPYR in human serum and plasma samples.
For detection of IPYR in human serum or plasma, a sandwich ELISA is developed.
For capture and detection of the antigen, aliquots of the anti-IPYR polyclonal
antibody (see Example 2) are conjugated with biotin and digoxygenin,
respectively.
Streptavidin-coated 96-well microwell plates are incubated with 100 l
biotinylated
anti-IPYR polyclonal antibody for 60 min at 10 g/ml in 10 mM phosphate, pH
7.4,
1% BSA, 0.9% NaCl and 0.1% Tween 20. After incubation, plates are washed three
times with 0.9% NaC1, 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 liquid samples obtained from patients. After binding of IPYR,
plates
are washed three times with 0.9% NaCI, 0.1% Tween 20. For specific detection
of
bound IPYR, wells are incubated with 100 l of digoxygenylated anti-IPYR
polyclonal antibody for 60 min at 10 g/ml in 10 mM phosphate, pH 7.4, 1% BSA,
0.9% NaCl 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 (Roche Diagnostics GmbH, Mannheim, Germany,
Catalog No. 1633716) for 60 min in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl
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 l ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog
No. 11685767) and OD is measured after 30-60 min at 405 nm with an ELISA
reader.
Example 5
ROC analysis to assess clinical utility in terms of diagnostic accuracy
Accuracy is assessed by analyzing individual liquid samples obtained from well-
characterized patient cohorts, i.e., 50 patients having undergone colonoscopy
and
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found to be free of adenoma or CRC, 50 patients diagnosed and staged as T;s-3,
NO,
MO of CRC, and 50 patients diagnosed with progressed CRC, having at least
tumor
infiltration in at least one proximal lymph node or more severe forms of
metastasis,
respectively. CEA as measured by a commercially available assay (Roche
Diagnostics, CEA-assay (Cat. No. 1 173 1629 for Elecsys Systems immunoassay
analyzer) and IPYR measured as described above are quantified in a serum
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 T15-3, NO, MO from healthy individuals for the combination of RCN1 with
the established marker CEA is calculated by regularized discriminant analysis
(Friedman, J.H., Regularized Discriminant Analysis, Journal of the American
Statistical Association 84 (1989) 165-175).
Preliminary data indicate that IPYR may also be very helpful in the follow-up
of
patients after surgery.
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