The present invention relates to the diagnosis of breast cancer.
It discloses the use of protein cellular retinoic acid binding protein
II in the diagnosis of breast cancer. It relates to a method for
diagnosis of breast cancer from a liquid sample, derived from an
individual by measuring cellular retinoic acid binding protein II
in said sample. Measurement of cellular retinoic acid binding protein
II can, e.g., be used in the early detection or diagnosis of breast
1. A method of detecting cellular retinoic acid-binding protein
II in a liquid biological sample, said method comprising the step
of: (a) providing a liquid sample obtained from an individual, (b)
contacting said sample with a specific binding agent for cellular
retinoic acid-binding protein II under conditions appropriate for
formation of a complex between said binding agent and the cellular
retinoic acid-binding protein II; and (c) detecting said complexes.
2. The method of claim 1 wherein the binding agent is an antibody.
3. The method of claim 1 wherein the binding agent comprises a
first and second antibody that each specifically bind to cellular
retinoic acid-binding protein II, wherein the first antibody is
a capture reagent that binds to cellular retinoic acid-binding protein
II and to a solid support, and the second antibody is labeled to
be directly or indirectly detectable.
4. The method of claim 1, wherein said sample is whole blood.
5. The method of claim 4, wherein said sample is plasma.
6. The method of claim 5, wherein said sample is serum.
7. The method of claim 1, wherein said sample comprises nipple
8. A method for diagnosing breast cancer comprising the steps of:
(a) providing a liquid sample obtained from an individual, (b) contacting
said sample with a specific binding agent for cellular retinoic
acid-binding protein II under conditions appropriate for formation
of a complex between said binding agent and cellular retinoic acid-binding
protein II, and (c) correlating the amount of complex formed in
(b) to the diagnosis of breast cancer.
9. The method of claim 8 wherein the binding agent is an antibody.
10. The method of claim 8 wherein the binding agent comprises a
first and second antibody wherein the first antibody is a capture
reagent that binds to the cellular retinoic acid-binding protein
II and to a solid support, and the second antibody is labeled to
be directly or indirectly detectable.
11. The method of claim 7, wherein said sample is serum.
12. The method of claim 7, wherein said sample is nipple aspirate
13. The method of claim 7 further comprising the step of contacting
said sample with a second specific binding agent that is specific
for a second breast cancer marker.
14. The method of claim 11 wherein the second marker is selected
form the group consisting of carcinoembryonic antigen (CEA) and
15. A method of monitoring the effectiveness of an anticancer therapy,
said method comprising: (a) determining pretreatment levels of cellular
retinoic acid-binding protein II in a biological sample recovered
from a patient, (b) administering an anticancer therapy to said
patient, and (c) determining the post treatment levels of cellular
retinoic acid-binding protein II in a biological sample obtained
from said patient.
16. The method of claim 15 wherein said biological sample is a
17. The method of claim 15, wherein said sample is serum or nipple
18. The method of claim 15 wherein the post treatment levels are
determined within a timeframe of about 3 hours to about 14 days.
19. The method of claim 15 wherein the post treatment levels are
determined within a timeframe of about 3 months to about 10 years.
20. An immunological kit comprising at least one specific binding
agent for cellular retinoic acid-binding protein II and auxiliary
reagents for measurement of cellular retinoic acid-binding protein
21. The immunological kit of claim 18 wherein the kit comprises
a first and second antibody that each specifically bind to cellular
retinoic acid-binding protein II, wherein the first antibody is
a capture reagent that binds to the cellular retinoic acid-binding
protein II and to a solid support, and the second antibody is labeled
to be directly or indirectly detectable.
 This application is a continuation of PCT/EP2004/006029
filed Jun. 4, 2004 and claims priority to European application EP
03012942.3 filed Jun. 6, 2003.
FIELD OF THE INVENTION
 The present invention relates to the diagnosis of breast
cancer. It discloses the use of cellular retinoic acid binding protein
II in the diagnosis of breast cancer. Furthermore, it especially
relates to a method for diagnosis of breast cancer from a liquid
sample, derived from an individual by measuring cellular retinoic
acid binding protein II in said sample. Measurement of cellular
retinoic acid binding protein II can, e.g., be used in the early
detection or diagnosis of breast cancer.
BACKGROUND OF THE INVENTION
 Cancer remains a major public health challenge despite progress
in detection and therapy. Amongst the various types of cancer, breast
cancer (BC) is one of the most frequent cancers among women in the
 The earlier cancer can be detected/diagnosed, the better
is the overall survival rate. This is especially true for BC. 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 BC as a public health problem, it is essential
that more effective screening and preventative measures for breast
cancer will be developed.
 The earliest detection procedures available at present for
breast cancer involve using clinical breast examination and mammography.
However, significant tumor size must typically exist before a tumor
is palpable or can be detected by a mammogram. The densitiy of the
breast tissue and the age are important predictors of the accuracy
of screening mammography. The sensitivity ranges from 63% in women
with extremely dense breasts to 87% in women with almost entirely
fatty breasts. The sensitivity increases with age from 69% in women
of about 40 years of age to 83% in women 80 years and older (Carney,
P. A., et al., Ann. Intern. Med. 138 (3) (2003) 168-175). Only 20-25%
of mammographically detected abnormalities that are biopsied prove
to be malignant. The visualization of precancerous and cancerous
lesions represents the best approach to early detection, but mammography
is an expensive test that requires great care and expertise both
to perform and in the interpretation of results (WHO, Screening
for Breast Cancer, May 10, 2002; Esserman, L., et al., J. Natl.
Cancer Inst. 94 (2002) 369-375).
 In the recent years a tremendous amount of so-called breast
specific or even so-called breast 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 breast (cancer) tissue versus a different
tissue or an adjacent normal tissue, respectively. Such approaches
may be summarized as differential mRNA display techniques.
 As an example for data available from mRNA-display techniques,
WO 00/60076 shall be mentioned and 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 BC. 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 mRNA may nonetheless
be hard to detect and find at all (Chen, G., et al., Molecular and
Cellular Proteomics, 1.4 (2002) 304-313). This lack of correlation
between mRNA-level and protein level is due to reasons like mRNA
stability, efficiency of translation, stability of the protein,
 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 BC. Wulfkuhle et al. Cancer
Research 62 (2002) 6740-6749 have identified fifty-seven proteins
which were differentially expressed between BC tissue and adjacent
normal tissue. No data from liquid samples obtained from an individual
 WO 02/23200 reports about twelve breast 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 BC as compared to sera obtained from healthy controls.
However, the identity of the molecule(s) comprised in such spot,
e.g their sequence, is not known.
 Nipple aspirate fluid (NAF) has been used for many years
as a potential non-invasive method to identify breast cancer-specific
markers. Kuerer et al. compared bilateral matched pair nipple aspirate
fluids from women with unilateral invasive breast carcinoma by 2D
gel electrophoresis (Kuerer, H. M., et al., Cancer 95 (2002) 2276-2282).
30 to 202 different protein spots were detected in the NAF of breasts
suffering from breast carcinoma and not in the matched NAF of the
healthy breasts. These spots were detected by a gel image analysis.
But the identity of the protein spots is not known.
 Despite the large and ever growing list of candidate protein
markers in the field of BC, 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, only diagnostic blood tests based on the detection
of cancer antigen 15-3 (CA 15-3), a tumor-associated mucin, and
carcinoembryonic antigen (CEA), a tumor associated glycoprotein,
are available to assist diagnosis in the field of BC. CA 15-3 is
usually increased in patients with advanced breast cancer. CA 15-3
levels are rarely elevated in women with early stage breast cancer
(Duffy, M. J., Critical Reviews in Clinical Laboratory Sciences
38 (2001) 225-262). Cancers of the ovary, lung and prostate may
also raise CA 15-3 levels. Elevated levels of CA 15-3 may be associated
with non-cancerous conditions, such as benign breast or ovary disease,
endometriosis, pelvic inflammatory disease, and hepatitis. Pregnancy
and lactation can also cause CA 15-3 levels to raise (National Cancer
Institute, Cancer Facts, Fact Sheet 5.18 (1998) 1-5). The primary
use of CEA is in monitoring colon cancer, especially when the disease
has metastasized. However, a variety of cancers can produce elevated
levels of CEA, including breast cancer.
 Due to the lack of organ and tumor specificity, neither
measurement of CA 15-3 nor measurement of CEA are recommended for
screening of BC. These tumor markers are helpful diagnostic tools
in follow-up care of BC patients (Untch, M., et al., J. Lab. Med.
25 (2001) 343-352).
 Whole blood, serum, plasma, or nipple aspirate fluid are
the most widely used sources of sample in clinical routine. The
identification of an early BC tumor marker that would allow 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 diagnosis of BC from blood. It is especially
important to improve the early diagnosis of BC, 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 BC diagnosis.
SUMMARY OF THE INVENTION
 Surprisingly, it has been found that use of the marker cellular
retinoic acid binding protein II 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 breast cancer comprising the steps of a) providing
a liquid sample obtained from an individual, b) contacting said
sample with a specific binding agent for cellular retinoic acid
binding protein II under conditions appropriate for formation of
a complex between said binding agent and cellular retinoic acid
binding protein II, and c) correlating the amount of complex formed
in (b) to the diagnosis of breast cancer
 Another preferred embodiment of the invention is a method
for the diagnosis of breast cancer comprising the steps of a) contacting
a liquid sample obtained from an individual with a specific binding
agent for cellular retinoic acid binding protein II under conditions
appropriate for formation of a complex between said binding agent
and cellular retinoic acid binding protein II, and b) correlating
the amount of complex formed in (a) to the diagnosis of breast cancer.
 As the skilled artisan will appreciate, any such diagnosis
is made in vitro. The patient sample is discarded afterwards. The
patient sample is merely used for the in vitro diagnostic 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
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 FIG. 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). The circle in the enlarged section
of these gels indicates the position for the protein cellular retinoic
acid binding protein II (CRABP-II). Using the same method this protein
has not been detected in healthy tissue.
 FIG. 2 FIG. 2 shows ROC-Curves: Breast Cancer versus Controls/Others
cancers. The x-axis indicates the value computed by subtracting
from 1 the specificity value. The y-axis indicates sensitivity.
In both the value of 1 corresponds to 100%. The ROC values for CRABP-II,
CEA and CA 15-3 have been determined to be 73%, 51%, and 54%, respectively.
 FIG. 3 FIG. 3 shows ROC-Curves: Breast Cancer versus Controls/Other
cancers excluding ovary cancer. The x-axis indicates the value computed
by subtracting from 1 the specificity value. The y-axis indicates
sensitivity. In both the value of 1 corresponds to 100%. The ROC
values for CRABP-II, CEA and CA 15-3 have been determined to be
74%, 50%, and 58%, respectively.
DESCRIPTION OF THE INVENTION
 The cellular retinoic acid binding protein II (CRABP-II)
(Swiss-PROT: P29373) is characterized by the sequence given in SEQ
ID NO: 1. This sequence translates to a molecular weight of 15,562
Da and to an isoelectric point at pH 5.43.
 The two isoforms of cellular retinoic acid binding proteins
(CRABP-I and -II) were first characterized by Siegenthaler et al.
1992. CRABP-II was shown to be the major isoform, highly expressed
in human epidermis by fibroblasts and keratinocytes (Siegenthaler,
G., Biochemical Journal 287 (1992) 383-389).
 An increased concentration of CRABP-II was found in keratoacanthoma
and squamous cell cancer but not in basal cell carcinoma of the
skin by Anders et al. (Anders, V., et al., Journal of Investigative
Dermatology 106 (1996) 1070-1074).
 In the cytoplasm, CRABP-II regulates the intracellular retinoic
acid (RA) concentration, transport, and metabolism. It has been
demonstrated that RA induced CRABP-II mRNA levels 2 fold in squamous
cell cancer by transcriptional upregulation (Vo, H. P., Crowe, D.
L., Anticancer Research 18 (1998) 217-224).
 The presence of CRABP-II in human breast cancer cells was
first described by Wang et al. 1998. They localized CRABP-II in
human breast cancer cells by immunohistochemistry (Wang, Y., et
al., Laboratory Investigation 78 (1998) 30 A).
 The function of CRABP-II in mammary carcinoma cells was
described by Budhu and Noy 2002 (Molecular and Cellular Biology
22 (2002) 2632-2641). The cytosolic CRABP-II undergoes a nuclear
localization upon binding RA and interacts with retinoic acid receptor
(RAR) by building a short lived CRABP-II--RAR-complex. The overexpression
of CRABP-II in MCF7 mammary cell lines enhances their sensitivity
to retinoic acid-induced growth inhibition (Budhu, A. S., Noy, N.,
 In a first proteomics analysis of matched normal ductal/lobular
units and ductal carcinoma in situ (DCIS) of the human breast Wulfkuhle
et al. (Cancer Research 62 (2002) 6740-6749) identified fifty-seven
proteins that were differentially expressed in normal and precancerous
cells. The level of CRABP-II was reported to be five times higher
in DCIS than in normal cells. A comparable increase has been reported
for as many as 23 proteins. But no further investigations were carried
out, e.g. whether CRABP-II could be detected in liquid samples (Wulfkuhle,
J. D. et al., supra).
 CRABP-II has been mentioned in different patent applications
besides a large number of genes and their proteins for diagnosing
or prognosing the development or progression of breast cancer (WO
02/77176, WO 02/101075, WO 02/59377). But the diagnostic application
has not been described.
 As obvious to the skilled artisan, the present invention
shall not be construed to be limited to the full-length protein
CRABP-II of SEQ ID NO:1. Physiological or artificial fragments of
CRABP-II, secondary modifications of CRABP-II, as well as allelic
variants of CRABP-II 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.
 In preferred embodiments, the novel marker CRABP-II may
be used for monitoring as well as for screening purposes.
 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 CRABP-II are directly correlated to tumor burden.
After chemotherapy a short term (few hours to 14 days) increase
in CRABP-II may serve as an indicator of tumor cell death. In the
follow-up of patients (from 3 months to 10 years) an increase of
CRABP-II can be used as an indicator for tumor recurrence.
 In a preferred embodiment the diagnostic method according
to the present invention is used for screening purposes. I.e., it
is used to assess subjects without a prior diagnosis of BC by measuring
the level of CRABP-II and correlating the level measured to the
presence or absence of BC.
 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) (Sobin, L. H., Wittekind, Ch. (eds): TNM Classification
of Malignant Tumours, fifth edition, 1997). The staging system for
breast cancer has recently been revised (Singletary, S. E., et al.,
Journal of Clinical Oncology 20 (2002) 3628-3636).
 What is especially important is, that early diagnosis of
BC translates to a much better prognosis. Therefore, best prognosis
have those patients as early as in stage T.sub.is, N0, M0 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 18% for patients diagnosed when distant metastases are already
 In the sense of the present invention early diagnosis of
BC refers to a diagnosis at a pre-cancerous state (DCIS) or at a
tumor stage where no metastases at all (neither proximal nor distal),
i.e., T.sub.is, N0, M0 or T1-4; N0; M0 are present. T.sub.is denotes
carcinoma in situ.
 In a preferred embodiment CRABP-II is used to diagnose BC
in a non metastatic stage, i.e., that diagnosis is made at stage
T.sub.is, N0, M0 or T1-3; N0; M0 (=T.sub.is-3; N0; M0).
 The diagnostic 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 CRABP-II is specifically measured
from this liquid sample by use of a specific binding agent.
 A specific binding agent is, e.g., a receptor for CRABP-II,
a lectin binding to CRABP-II or an antibody to CRABP-II. A specific
binding agent has at least an affinity of 10.sup.7 l/mol for its
corresponding target molecule. The specific binding agent preferably
has an affinity of 10.sup.8 l/mol or even more preferred of 10.sup.9
l/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 CRABP-II. 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 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 CRABP-II. 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 also 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
monoclonal and polyclonal antibodies have been used. Polyclonal
antibodies have been raised in rabbits. However, clearly also polyclonal
antibodies from different species, e.g. rats or guinea pigs can
also be used. Monoclonal antibodies have been produced using spleen
cells from immunized mice. 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 CRABP-II in a method
according to the present invention is yet another preferred embodiment.
 As the skilled artisan will appreciate now, that CRABP-II
has been identified as a marker which is useful in the diagnosis
of BC, 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 CRABP-II for immunization. Preferably, a synthetic peptide comprises
a subsequence of SEQ ID NO:1 which is specific for CRABP-II, i.e.,
which has a comparatively low homology to other/related polypeptides.
It is preferred that the synthetic peptide comprises a contiguous
subsequence consisting of 5 to 25 amino acid residues of SEQ ID
NO:1. More preferred, the peptide comprises a contiguous subsequence
consisting of 10 to 15 amino acid residues of SEQ ID NO:1.
 A very preferred subsequence of CRAP-II consists of the
amino acid residues 85-96 of SEQ ID NO:1. A further preferred subsequence
consists of the amino acid residues 106-120 of SEQ ID NO:1 and Cysteine
residue added to its C-terminus for a facilitated coupling via SH-chemistry.
 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 CRABP-II under
conditions appropriate for formation of a binding agent CRABP-II-complex.
Such conditions need not be specified, since the skilled artisan
without any inventive effort can easily identify such appropriate
 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 BC. As the skilled artisan will appreciate there
are numerous methods to measure the amount of specific binding agent
CRABP-II-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 CRABP-II is detected in a sandwich type assay
format. In such assay a first specific binding agent is used to
capture CRABP-II 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
CRABP-II can be measured from a liquid sample obtained from an individual
sample. No tissue and no biopsy sample is required to apply the
marker CRABP-II in the diagnosis of BC.
 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
 In a further preferred embodiment the method according to
the present invention is practiced with whole blood as liquid sample
 In a further preferred embodiment the method according to
the present invention is practiced with nipple aspirate fluid as
liquid sample material.
 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 CRABP-II in a bodily fluid
sample. Even more surprising they have been able to demonstrate
that the presence of CRABP-II in such liquid sample obtained from
an individual can be correlated to the diagnosis of breast cancer.
 Antibodies to CRABP-II with great advantage can be used
in established procedures, e.g., to detect breast cancer cells in
situ, in biopsies, or in immunohistological procedures.
 Preferably, an antibody to CRABP-II is used in a qualitative
(CRABP-II present or absent) or quantitative (CRABP-II amount is
 Measuring the level of protein CRABP-II has proven very
advantageous in the field of BC. Therefore, in a further preferred
embodiment, the present invention relates to use of protein CRABP-II
as a marker molecule in the diagnosis of breast cancer from a liquid
sample obtained from an individual.
 The term marker molecule is used to indicate that an increased
level of the analyte CRABP-II as measured from a bodily fluid of
an individual marks the presence of BC.
 It is especially preferred to use the novel marker CRABP-II
in the early diagnosis of breast cancer.
 The use of protein CRABP-II itself, represents a significant
progress to the challenging field of BC diagnosis. Combining measurements
of CRABP-II with other known markers, e.g. CA 15-3 and CEA, or with
other markers of BC presently known or yet to be discovered, leads
to further improvements. Therefore in a further preferred embodiment
the present invention relates to the use of CRABP-II as a marker
molecule for breast cancer in combination with one or more marker
molecules for breast cancer in the diagnosis of breast cancer from
a liquid sample obtained from an individual. In this regard, the
expression "one or more" denotes 1 to 10, preferably 1
to 5, more preferred 3. Preferred selected other BC markers with
which the measurement of CRABP-II may be combined are CEA and CA
15-3. Most preferred, CRABP-II is used as part of a marker panel
at least comprising CRABP-II and CA 15-3. Thus, a further preferred
embodiment of the present invention is the use of the protein CRABP-II
as a marker molecule for breast cancer in combination with one or
more marker molecules for breast cancer in the diagnosis of breast
cancer from a liquid sample obtained from an individual, whereby
the at least one other marker molecule is CA 15-3.
 Preferably, the inventive method is used with samples of
patients suspected of suffering from breast cancer. An individual
suspected of suffering from breast cancer is an individual for which
other types of cancers have been excluded. Other cancers include
but are not limited to cancers of the colon, lung, stomach, ovary,
and prostate. A preferred embodiment of the invention is therefore
a method for the diagnosis of breast cancer comprising the steps
of a) providing a liquid sample obtained from an individual suspected
of suffering from breast cancer, b) contacting said sample with
a specific binding agent for cellular retinoic acid binding protein
II under conditions appropriate for formation of a complex between
said binding agent and cellular retinoic acid binding protein II,
and c) correlating the amount of complex formed in (b) to the diagnosis
of breast cancer.
 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 CRABP-II and auxiliary reagents for measurement
 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.degree. diagonal line from the lower left corner to the
upper right corner. Most plots fall in between these two extremes.
(If the ROC plot falls completely below the 45.degree. 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.gtoreq.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
 Clinical utility of the novel marker CRABP-II has been assessed
in comparison to and in combination with the established marker
CA 15-3 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 50
samples each from patients with invasive ductal or lobular carcinoma
in T1-3; N0; M0, more progressed tumor, i.e., T4 and/or various
severity of metastasis (N+ and/or M+), medullary, papillary, mucinous
and tubular carcinoma, ductal carcinoma in situ, and healthy controls,
 The diagnostic method based on measurement of CRABP-II alone
in comparison to the established marker CA 15-3 alone has been found
to have an at least as good a diagnostic accuracy (sensitivity/specificity
profile) as demonstrated by the area under the curve.
 The examples, 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. TABLE-US-00001 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 iodacetamid 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 UICC International Union Against Cancer
Identification of Cellular Retinoic Acid Binding Protein II (CRABP-II)
as a Potential Breast Cancer Marker
Sources of Tissue
 In order to identify tumor-specific proteins as potential
diagnostic markers for breast cancer, analysis of two different
kinds of tissue is performed using proteomics methods.
 In total, tissue specimen from 14 patients suffering from
breast cancer are analyzed. From each patient two different tissue
types are collected from therapeutic resections: Tumor tissue (>80%
tumor) (T), and adjacent healthy tissue (N). The latter tissue type
serves as matched healthy control sample. Tissues are immediately
snap frozen after resection and stored at -80.degree. C. before
processing. Tumors are diagnosed by histopathological criteria.
 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 MgCl.sub.2, 1% Genapol X-080, 0.02% Na-azide, Complete.RTM.
EDTA-free [Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 1
873 580]) and subsequently homogenized in a Wheaton.RTM. glass homogenizer
(20.times.loose fitting, 20.times.tight fitting). 3 ml of the homogenate
are subjected to a sucrose-density centrifugation (10-60% sucrose)
for 1 h at 4,500.times.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.
Immobilization of Monoclonal Antibody Anti-Human Albumin on CNBr-Activated
 Freeze-dried CNBr-activated Sepharose 4B (Amersham Biosciences,
17-0430-01) is reswollen and washed according to the instructions
of the manufacturer. Monoclonal antibody directed against human
albumin is dissolved in 0.1 M NaHCO.sub.3, pH 8.3, 0.5 M NaCl, 10
mg/ml. One ml antibody solution is mixed with 1 ml reswollen CNBr-activated
Sepharose 4B. The reaction time is 1 h. Blocking of the remaining
acitve groups and washing of the gel is carried out according to
the instructions of the manufacturer.
Depletion of Serum Albumin
 7 ml anti-albumin gel is equilibrated in lysis buffer without
Genapol X-080. 7 ml of the upper fraction of the sucrose-density
centrifugation (see above, tissue preparation) are applied onto
the column and washed through with lysis buffer without Genalpol
X-080. The combined effluent is used for the isoelectric focussing
Isoelectric Focussing (IEF) and SDS-PAGE
 For IEF, 3 ml of the HSA-depleted tissue preparation 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.RTM. Ultra-15 device (Millipore GmbH,
Schwalbach, Germany) and the protein concentration is determined
using the Bio-Rad.RTM. 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 1.5 mg of
protein sample buffer is added to a final volume of 350 .mu.l. 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 3500 V; (3.)
22 h at constant 3500 V giving rise to 82 kVh. After IEF, strips
are stored at -80.degree. C. or directly used for SDS-PAGE.
 Prior to SDS-PAGE the strips are incubated in equilibration
buffer (6 M urea, 50 mM Tris/HCl, pH 8.8, 30% glycerol, 2% SDS),
for reduction DTT (15 min, +50 mg DTT/10 ml), and for alkylation
LAA (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 and thereafter 1 h at 17 W/gel. Subsequently, the gels
are fixed (50% methanol, 10% acetate) and stained overnight with
Novex.TM. Colloidal Blue Staining Kit (Invitrogen, Karlsruhe, Germany,
Cat No. LC6025, 45-7101)
Detection of CRABP-II as a Potential Marker for Breast Cancer
 Each patient is analyzed separately by image analysis with
the Proteome Weaver.RTM. software (Definiens AG, Germany, Munchen).
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 (Ultraflex.TM. Tof/Tof, Bruker Daltonik GmbH,
Bremen, Germany). For each patient, 4 gels from the tumor sample
are compared with 4 gels each from adjacent tissue and analyzed
for distinctive spots corresponding to differentially expressed
proteins. By this means, protein CRABP-II is found to be specifically
expressed or strongly overexpressed in tumor tissue and not detectable
in healthy control tissue. It therefore--amongst many other proteins--qualifies
as a candidate marker for use in the diagnosis of breast cancer.
Generation of Antibodies to the Breast Cancer Marker Protein CRABP-II
 Polyclonal antibody to the breast cancer marker protein
CRABP-II is generated for further use of the antibody in the measurement
of serum and plasma and blood levels of CRABP-II by immunodetection
assays, e.g. Western Blotting and ELISA
Recombinant Protein Expression and Purification
 In order to generate antibodies to CRABP-II, 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 for in-vitro transcription
and expression of the nucleotide sequence coding for the CRABP-II
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.RTM. 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 1 l batch for protein expression.
 Purification of His-CRABP-II fusion protein is done following
standard procedures on a Ni-chelate column. Briefly, 1 l of bacteria
culture containing the expression vector for the His-CRABP-II 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.RTM.. 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.
 In addition, CRABP-II protein is expressed and purified
as described in Kleywegt, G. J. et al., Structure 2 (1994) 1241-1258,
particularly page 1252 ("Protein preparation").
 Peptides are synthesized and purified by means of state
of the art chemistry.
 The CRABP-II-peptide corresponding to positions 85-96 of
SEQ ID NO:1 contains a cysteine residue. The peptide is furtheron
also referred to as CRABP-II (85-96) or the CRABP-II (85-96) peptide.
 To the CRABP-II-peptide corresponding to positions 106-120
of SEQ ID NO:1 a Cysteine residue is added at the C-terminus of
the peptide. The peptide is furtheron also referred to as CRABP-II
(106-120 Cys) or the CRABP-II (106-120 Cys) peptide.
Synthesis of Hemocyanin-Peptide Conjugates for the Generation of
 Synthesis is carried out using heterobifunctional chemistry
 The CRABP-II (85-96) peptide is coupled to 3-maleimidohexanoyl-N-hydroxysuccinimidester
(MHS) activated hemocyanin. Similarly, the CRABP-II (106-120 Cys)
peptide is coupled to 3-maleimidohexanoyl-N-hydroxysuccinimidester
(MHS) activated hemocyanin. Hemocyanin is brought to 10 mg/ml in
100 mM NaH.sub.2PO.sub.4/NaOH, pH 7.2. Per ml hemocyanin 100 .mu.l
MHS (12.3 mg in DMSO) are added and incubated for 1 h. The sample
is dialyzed over night against 100 mM NaH.sub.2PO.sub.4/NaOH, pH
6.5 and adjusted to 6 mg/ml with dialysis buffer. The CRABP-II (85-96)
peptide or the CRABP-II (106-120 Cys) peptide is dissolved in DMSO
(5 mg/ml for a peptide of 1,500 Da [Dalton]). Per ml MHS-activated
hemocyanin (6 mg/ml) 20 .mu.l of 100 mM EDTA, pH 7.0 and 100 .mu.l
of the cysteine-containing CRABP-II-peptide (85-96) or the CRABP-II
(106-120 Cys) peptide are added. After 1 h the remaining maleimide
groups are blocked by the addition of 10 .mu.l 0.5 M cysteine/HCl
per ml reaction mixture. This preparation is used for immunization
without further purification.
Production of Monoclonal Antibodies Against the CRABP-II
 a) Immunization of Mice
 12 week old A/J mice are initially immunized intraperitoneally
with 100 .mu.g CRABP-II or hemocyanin-peptide-conjugate (see above).
This is followed after 6 weeks by two further intraperitoneal immunizations
at monthly intervals. In this process each mouse is administered
100 .mu.g CRABP-II or hemocyanin-peptide-conjugate adsorbed to aluminium
hydroxide and 10.sup.9 germs of Bordetella pertussis. Subsequently
the last two immunizations are carried out intravenously on the
3rd and 2nd day before fusion using 100 .mu.g CRABP-II or hemocyanin-peptide-conjugate
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.times.10.sup.8
spleen cells of the immunized mouse are mixed with 2.times.10.sup.7
myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10
min at 300.times.g and 4.degree. C.). The cells are then washed
once with RPMI 1640 medium without foetal calf serum (FCS) and centrifuged
again at 400.times.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.degree. 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.times.g and 4.degree. C. The sedimented cells
are taken up in RPMI 1640 medium containing 10% FCS and sown in
hypoxanthine-azaserine selection medium (100 mmol/l hypoxanthine,
1 .mu.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. CRABP-II-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 1.times.10.sup.5
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 .mu.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 in Enzymology 121 (1986) 587-695).
Generation of Polyclonal Antibody:
 For immunization, a fresh emulsion of the protein solution
(100 .mu.g/ml CRABP-II or hemocyanin-peptide-conjugate comprising
the CRABP-II (85-96) peptide or the CRABP-II (106-120 Cys) peptide)
and complete Freund's adjuvant at the ratio of 1:1 is prepared.
With respect to each peptide, each rabbit is immunized with 1 ml
of the emulsion of the at days 1, 7, 14 and 30, 60 and 90. Blood
is drawn and resulting anti-CRABP-II 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 .mu.l/ml of diluted sample) is added
drop-wise under vigorous stirring. After 30 min the sample is centrifuged
(13,000.times.g, 30 min, 4.degree. 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 .mu.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.times.g, 15 min, 4.degree.
 The supernatant is discarded. The pellet is dissolved in
10 mM NaH.sub.2PO.sub.4/NaOH, pH 7.5, 30 mM NaCl and exhaustively
dialyzed. The dialysate is centrifuged (13,000.times.g, 15 min,
4.degree. C.) and filtered (0.2 .mu.m).
Biotinylation of Polyclonal Rabbit IgG
 Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH.sub.2PO.sub.4/NaOH,
pH 7.5, 30 mM NaCl. Per ml IgG solution 50 .mu.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 NaH.sub.2PO.sub.4/NaOH,
pH 7.5, 30 mM NaCl). The fraction containing biotinylated IgG are
collected. Monoclonal antibodies are biotinylated according to the
Digoxygenylation of Polyclonal Rabbit IgG
 Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH.sub.2PO.sub.4/NaOH,
30 mM NaCl, pH 7.5. Per ml IgG solution 50 .mu.l digoxigenin-3-O-methylcarbonyl-.epsilon.-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.RTM.
200 (10 mM NaH.sub.2PO.sub.4/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.
Western Blot for the Detection of CRABP-II in Human Serum and Plasma
 SDS-PAGE and Western Blotting are carried out using reagents
and equipment of Invitrogen, Karlsruhe, Germany. Human plasma samples
are diluted 1:20 in reducing NuPAGE.RTM. (Invitrogen) LDS sample
buffer and heated for 5 min at 95.degree. C. 10 .mu.l aliquots are
run on 4-12% NuPAGE.RTM. gels (Bis-Tris) in the MES running buffer
system. The gel-separated protein mixture is blotted onto nitrocellulose
membranes using the Invitrogen XCell II.TM. Blot Module (Invitrogen)
and the NuPAGE.RTM. transfer buffer system. The membranes are washed
3 times in PBS/0.05% Tween-20 and blocked with SuperBlock Blocking
Buffer (Pierce Biotechnology, Inc., Rockford, Ill., USA). The biotinylated
primary antibody is diluted in SuperBlock Blocking Buffer (0.01-0.2
.mu.g/ml) and incubated with the membrane for 1 h. The membranes
are washed 3 times in PBS/0.05% Tween-20. The specifically bound
biotinylated primary antibody is labeled with a streptavidin-HRP-conjugate
(20 mU.sub.ABTS/ml in SuperBlock Blocking Buffer). After incubation
for 1 h, the membranes are washed 3 times in PBS/0.05% Tween-20.
The bound streptavidin-HRP-conjugate is detected using a chemiluminescent
substrate (SuperSignal West Femto Substrate, Pierce Biotechnology,
Inc., Rockford, Ill., USA) and autoradiographic film. Exposure times
varies from 10 min to over night.
ELISA for the Measurement of CRABP-II in Human Serum and Plasma
 For detection of CRABP-II in human serum or plasma, a sandwich
ELISA was developed using streptavidin-coated 96-well microtiter
 For detection of CRABP-II in human serum or plasma, a sandwich
ELISA was developed using streptavidin-coated 96-well microtiter
 Twenty microliter of a human serum or plasma sample or a
serial dilution of the recombinant CRABP-II protein as standard
antigen were incubated with 100 .mu.l biotinylated polyclonal anti-CRABP-II
(85-96) antibody (0.1 .mu.g/ml) and with digoxygenylated monoclonal
anti-CRABP-II (0.1 .mu.g/ml) in 10 mM phosphate, pH 7.4, 1% BSA,
0.9% NaCl and 0.1% Tween-20. After incubation over night at room
temperature, the plates were washed three times with 0.9% NaCl,
0.1% Tween-20. For the detection of antigen-antibody complexes,
100 .mu.l of an monoclonal anti-digoxigenin peroxidase conjugate
in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20
were added and incubated for 2 h. The excess of conjugate was removed
by washing the plates three times with 0.9% NaCl , 0.1% Tween-20.
The amount of bound conjugate was detected by incubation with 100
.mu.l ABTS solution (Roche Diagnostics GmbH, Penzberg, Germany,
Catalog No. 11685767) for 30-60 min. The color development was quantified
at 405 nm using a ELISA reader. The concentration of CRABP-II in
a serum or plasma sample was calculated from the standard curve
using a serial dilution of recombinant CRABP-II.
Marker Evaluation, Sensitivity and Specificity; 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. The control collective
(see Table 1) contains 50 patients having undergone mammography.
40 patients are found mammography negative and no symptoms of other
breast diseases are detected. 5 patients are diagnosed with mastitis
(3 of 5 are mammography positive) and 5 patients are diagnosed with
microcalcification (all 5 are mammography positive). The sample
cohort is summarized in Table 1. TABLE-US-00002 TABLE 1 Healthy
patients 50 .SIGMA. Healthy controls 40 (mammography negative) Mastitis
5 Mammography 3 positive Microcalcification 5 Mammography 5 positive
Breast cancer .SIGMA. Stage 50 UICC I 20 UICC II 19 UICC III 10
UICC IV 1 Other cancers 120 Colon cancer 40 Lung cancer 20 Stomach
cancer 20 Ovary cancer 20 Prostate cancer 20
 The 50 breast cancer patients were put together from patients
with invasive ductal and invasive lobular carcinomas of different
stages. Due to the aim to diagnose breast cancer at early stages,
the proportion of UICC I and UICC II stages was 78%. To analyze
the specificity to other solid tumors a collective of 40 colon,
20 lung, 20 stomach, 20 ovary and 20 prostate cancer samples were
also measured. CA 15-3 and CEA as measured by commercially available
assays (Roche Diagnostics, CA 15-3-assay: Cat. No. 0 304 5838 and
CEA-assay: Cat. No. 1731629) for Elecsys.RTM. Systems immunoassay
analyzer) and CRABP-II measured as described above have been quantified
in a serum obtained from each of these individuals.
 The cut-off is defined with respect to the 95% percentile
of the control group, equalling 95% specificity. Thus, in the present
series of experiments the Cut-off value for CRABP-II is set to 0.92
 It is noted that the 3 positive (assumed to be false-positive)
control patients of the CRABP-II measurement in the "healthy
control" group are no mastitis or microcalcification patients.
Such patients usually give rise to a high rate of positive results
based on mammography. It is found that there is no positive reaction
in the lung, stomach and prostate cancer group. There are only three
positive results in colon cancer patients. This observation is comparable
to that of the healthy control group. The most positive reactions
are observed in the ovarian cancer group, that is to say 6 out of
 The data summarizing sensitivity and specificity of CRABP-II
in comparison to the markers CEA and CA 15-3 are given in Table
2 and Table 3. TABLE-US-00003 TABLE 2 Sensitivity Number of positive
results CRABP-II CA 15-3 CEA UICC I 4/20 1/20 1/20 UICC II 5/19
4/19 4/19 UICC III 2/10 6/10 3/10 UICC IV 1/1 1/1 1/1 Total 12/50
12/50 9/50 Sensitivity 24% 24% 18%
 TABLE-US-00004 TABLE 3 Specificity Values given in [%] CRABP-II
CA 15-3 CEA Controls 94 90 90 Other cancers: 96 90 61 Colon + lung
+ stomach Colon + lung + stomach + 91 81 64 ovary Colon + lung +
stomach + 93 79 68 ovary + prostate All controls 93 82 75
 ROC-analysis was performed according to Zweig, M. H., and
Campbell, supra. Discriminatory power for differentiating patients
in the breast cancer group from the "healthy" control
group as measured by the area under the curve was found to be at
least as good for CRABP-II (64%) as compared to the established
markers CA 15-3 (60%) and CEA (65%), respectively. On the other
hand CRABP-II showed a high specificity for breast cancer, since
there was no positive result in all stomach, lung and prostate cancer
samples. In colon cancer only 3 out of 40 samples were positive
(comparable to the healthy control group) and in ovary cancer 6
out of 20 were found positive. This leads to an improved discriminating
power of CRABP-II (73%) compared to CA 15-3 (54%) and CEA (51%),
if the breast cancer collective is compared with all controls including
all other solid tumors. TABLE-US-00005 TABLE 4 ROC values Values
given in [%] CRABP-II CA 15-3 CEA Breast cancer/controls 64 60 65
Breast cancer/controls + other cancers 73 54 51
 In conclusion, CRABP-II is as sensitive as CA 15-3 and at
the same time displays a higher specificity in the enlarged control
group, i.e. in the control group including other cancers. Furthermore,
CRABP-II detects more tumors at early stages. CRABP-II is highly
specific for breast tumors. No positive results have been obtained
with serum samples obtained from patients with lung, stomach and
prostate cancer and only minor positive reactions with samples obtained
from patients with colon cancer. In ovary cancer samples the specificity
is lower, but still higher than the specificity of the marker CA
15-3. Using all control samples including all other solid tumors
the discriminative power of CRABP-II (73%) is higher than for CA
15-3 (54%) and CEA (51%). The data indicate that CRABP-II may also
be very helpful in the diagnosis of breast cancer or in the follow-up
of patients after surgery.
 In some of the samples from BC patients both the levels
of CRABP-II as well as the level of CA 15-3 are elevated. In addition,
either CRABP-II or CA 15-3 is positive in individual samples obtained
from different breast cancer patients. This leads to a higher sensitivity
if both markers are measured in a patient sample. If a patient sample
is classified as positive in case one of the markers CRABP-II or
CA 15-3 is positive, then a sensitivity of 40% is achieved. Due
to the high specificity of CRABP-II, the specifitity of this combination
is comparable to the specificity of CA 15-3 alone. Thus the increased
sensitivity of the marker combination CRABP-II and CA 15 -3 does
not go to the expense of specificity (CA 15-3 alone 75% and the
combination 78%). TABLE-US-00006 TABLE 5 Sensitivity Number of positive
results CRABP-II and/or CA 15-3 UICC I 5/20 UICC II 7/20 UICC III
7/10 UICC IV 1/1 Total 20/50 Sensitivity 40%
 TABLE-US-00007 TABLE 6 Specificity Values given in [%] CRABP-II
and/or CA 15-3 Controls 86 Other cancers: 86 Colon + lung + stomach
Colon + lung + stomach + ovary 77 Colon + lung + stomach + ovary
+ prostate 76 All controls 78