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Cancer Patent Abstract
The present invention is directed to methods of identifying women
at increased risk of having ovarian cancer. Identification is based
upon the extent to which biological samples derived from the women
exhibit elevated levels of eosinophil-derived neurotoxin (EDN).
Cancer Patent Claims
What is claimed is:
1. A method of determining whether a human female subject is at
increased risk of having ovarian cancer relative to the general
population, comprising: (a) obtaining a test biological sample of
urine from said subject; (b) determining the amount of eosinophil-derived
neurotoxin (EDN) in said test biological sample wherein said EDN
is characterized by the amino acid sequence of SEQ ID NO:1; (c)
comparing the amount of EDN determined in step (b) with the amount
in one or more control biological samples of urine; and (d) concluding
that said subject is at increased risk of having ovarian cancer
relative to the general population if the amount of EDN in said
test biological sample is at least 20% higher than in said control
biological samples.
2. The method of claim 1, wherein the determination of EDN amount
is accomplished using an immunoassay.
3. The method of claim 1, wherein the amount of EDN is determined
by mass spectrometry.
4. The method of claim 1, wherein the amount of EDN is determined
by surface enhanced laser desorption/ionization mass spectrometry.
5. The method of any one of claims 1 or 2-4, wherein said subject
is selected for testing based upon a clinical determination that
she is at an elevated risk of having or developing ovarian cancer.
Cancer Patent Description
FIELD OF THE INVENTION
The present invention is in the field of tumor cell markers and
is particularly concerned with methods of detecting cancer by assaying
samples for eosinophil-derived neurotoxin (EDN). In its most preferred
embodiment, the invention is directed to methods in which urine
samples obtained from a woman are assayed for EDN to determine her
risk of either having or developing ovarian cancer.
BACKGROUND OF THE INVENTION
Approximately 70% of women with ovarian cancer are not diagnosed
until the disease has reached an advanced stage, i.e., until it
has spread to the upper abdomen (stage III) or beyond (stage IV).
The five-year survival rate for such women is only about 15%-20%.
In contrast, the five-year survival rate for patients diagnosed
with stage I disease is close to 90%. Thus, an assay for screening
women for early stage disease would be of great benefit.
Unfortunately, the diagnostic tests presently available for ovarian
cancer are not well suited to patient screening. For example, serum
levels of the tumor marker CA 125 are elevated in the majority of
women with stage III or stage IV cancer, but in less than half of
the women with stage I disease. In addition, there are many factors
unrelated to ovarian cancer that also result in elevated serum levels
of CA 125 and which can produce false positives. Attempts to use
other assays, either alone or in combination with the CA 125 test,
have met with only limited success.
In the late 1970's, Scheid proposed that serum RNase activity might
be correlated with ovarian cancer (Scheid, et al., Cancer 39:2204
(1977)). Subsequent studies confirmed this relationship and it was
suggested that RNase activity could be used as a diagnostic tool,
either alone or in conjunction with the CA 125 test (Schleich, et
al., Eur. J Gynaec. Oncol. 7:76-81 (1986); Schleich, et al., Cancer
Res. Clin. Oncol. 113:603-607 (1987)). However, one problem with
using total RNase activity as a diagnostic marker is that the levels
of this enzyme tend to be elevated in many non-cancerous conditions.
As a result, attempts have been made to identify particular RNase
enzymes that are more specific for cancer (see, e.g., U.S. Pat.
No. 5,866,119).
An RNase enzyme that appears to play an important role in inflammatory
and allergic diseases is eosinophil-derived neurotoxin (EDN). The
sequence for both the human EDN gene and protein have been reported
and comparisons have been made with other ribonucleases (Rosenberg,
et al., Proc. Nat'l Acad. Sci. USA 86:4460-4464 (1989); Hamann,
et al., Gene 83:162-167 (1989); Hamann, et al., Genomics 7:535-546
(1990); Barker, et al., J. Immunol. 143:952-955-(1989);and Beintema,
et al., Biochemistry 27:4530-4533 (1988)). Immunoassays for EDN
have been developed and used in the diagnosis of inflammatory bowel
disease (U.S. Pat. No. 5,928,883). In addition, an ELISA kit for
measuring human EDN is commercially available (Medical and Biological
Laboratories Co., Ltd., Watertown, Mass.). Although this enzyme
has been extensively studied, there do not appear to have been any
reports suggesting that it may be used in helping to diagnose ovarian
cancer.
SUMMARY OF THE INVENTION
The present invention is based upon experiments in which surface
enhanced laser desorption/ionization (SELDI) mass spectrometry was
used to examine urine specimens from women diagnosed as having ovarian
cancer. The results were compared with those derived from urine
specimens from women with either benign gynecological disease or
from subjects without known disease. Using this technique, a protein
was identified that was elevated in 60% of patients with ovarian
cancer, but in only 29% of patients with non-cancerous ovarian disease
and in 18% of disease-free individuals. Sequence analysis was then
performed and the protein was identified as EDN.
In its first aspect, the invention is directed to a method of screening
a human female subject for the presence of an abnormal ovarian growth
(e.g., a benign or malignant tumor) and to therefore determine whether
she is at increased risk of having ovarian cancer relative to the
general population. This is accomplished by removing a biological
sample from the subject and then assaying it to determine the amount
of EDN present. The results obtained are compared with those from
control samples derived from either the general population or from
subjects believed to be free of ovarian cancer. A conclusion is
drawn that the test subject has, or is at a high risk of developing,
ovarian cancer if the amount of EDN in the test biological sample
is significantly higher than the amount in the control samples.
Standard clinical methods may be used for obtaining biological
samples with the most preferred sample being urine. Alternatively,
blood or plasma may be used in assays or assays may be performed
on fluid or tissue derived from the ovary of a patient. Controls
may be selected using methods that are well known in the art. Once
a level has become well established for the control population,
assay results from test biological samples can be directly compared
with these known levels. Women identified as being at increased
risk would then undergo further clinical evaluation to confirm that
an abnormal ovarian growth is present and to determine whether the
growth is malignant or benign.
Any method for assaying EDN levels is compatible with the present
invention. However, the most preferred method is immunoassay, e.g.,
a radioimmunoassay or an ELISA. As discussed further in the Examples
section, SELDI mass spectrometry can also be used to determine whether
EDN levels are elevated. A "significantly elevated" amount
of EDN, for the purposes of the present invention, means that the
concentration in the test biological sample is higher than the concentration
in the control population to a degree that is statistically significant
using standard scientific criteria. In general, a test subject would
be considered as being at "increased risk" of having or
developing ovarian cancer if the amount of EDN present in the test
biological sample is at least 20% higher than that seen in the control
samples and the likelihood would increase as the difference became
more pronounced, e.g., as it rose above 40%, 60% or 80%. Of particular
concern would be women showing an elevation of fivefold, tenfold
or more.
As discussed above, assays of EDN levels provide a good indication
of whether a female subject has a benign or malignant growth of
the ovary. One way of distinguishing patients with malignant growths
from those with benign growths is based upon the form of EDN present.
Specifically, EDN in patients with ovarian cancer is more heavily
dimerized and the dimers are more heavily glycosylated than in patients
with benign growths. Thus, by comparing the amount of EDN dimer
present in a sample to the amount of monomer present, a determination
can be made as to whether a subject has a benign or a malignant
growth. If quantitation is based on protein, then a ratio of D.sub.p/M.sub.p,
where Dp=EDN dimer protein and Mp=EDN monomer protein, of less than
about 2 is indicative of a subject with a benign growth and a ratio
of 2 or greater is indicative of a patient with a malignant growth.
If quantitation is based upon glycosylation, then a ratio of Dg/Mg,
where Dg=glycosylation associated with EDN dimer and Mg=glycosylation
associated with EDN monomer, of about 0.5-1.5 would be indicative
of a benign growth. A ration of greater than 3 would indicate the
presence of a malignancy. Any method for evaluating dimer and monomer
levels is compatible with the invention. One method that has been
found to be effective is to immune precipitate EDN from a sample,
separate proteins by electrophoresis, perform a western blot and
then stain for either protein or glycosylation. Monomeric EDN should
migrate in gels with an apparent molecular weight of about 17 kDa
and dimers with an apparent molecular weight of about 35 kDa.
It should also be possible to distinguish between a sample from
a patient with a benign growth and a sample from a patient with
a malignant growth based upon the total amount of glycosylation
associated with EDN, i.e., glycosylation associated with all forms
of EDN combined. Specifically, EDN glycosylation in a sample from
a patient with ovarian cancer should be significantly greater than
in a control sample or group of sample derived from one or more
patients with a benign ovarian growth. The term "significantly
greater" means greater to a degree that is statistically significant
using accepted standards of analysis in the art. For example, samples
derived from patients with ovarian malignancies might have a total
amount of glycosylation that is at least 20% or 40% greater than
that in samples derived from patients with benign growths. The main
advantage of this method is that it eliminates the need to separate
dimeric forms of EDN from monomeric forms.
Although the EDN assays discussed above may be used alone as a
screening tool for ovarian cancer, these assays may also be combined
with other diagnostic tests for ovarian cancer. In particular, the
CA 125 assay may be performed in conjunction with the EDN assay.
Patients showing both elevated EDN levels and elevated CA 125 levels
would need to be further examined for the presence of tumors and/or
closely monitored for signs of neoplastic disease. The EDN assay
may also be used as part of a more comprehensive screening procedure
in which assays for one or more markers of other cancers or for
other diseases are performed. Markers evaluated may include prostate
specific antigen, BRCA-1 or BRCA-2.
In addition to using EDN assays as part of a general screening
procedure, the assays may also be used more selectively to monitor
women already identified as being at high risk for developing ovarian
cancer, for example, women in which ovarian cancer is very prevalent
in their family history.
Although EDN is good marker for ovarian cancer, it is expected
that other types of cancer will also correlate with elevated levels
of this protein. Such cancers include, but are not limited to: adenocarcinoma;
leukemia; lymphoma; melanoma; myeloma; sarcoma; and teratocarcinoma.
Particular organs affected may include the adrenal gland, bladder,
bone, bone marrow, brain, breast, cervix, gall bladder, colon, stomach,
heart, kidney, liver, lung, muscle, pancreas, parathyroid, prostate,
thyroid and uterus. In each case, the same biological assays discussed
above would be applied to identify patients with an elevated risk
of having or developing the disease.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: EDN amino acid sequence. FIG. 1 shows the amino acid sequence
of human EDN (SEQ ID NO: 1). As discussed herein, this protein has
been found to be elevated in patients with ovarian cancer.
FIG. 2: EDN gene sequence. FIG. 2 shows the nucleotide sequence
of the human gene coding for EDN (SEQ ID NO:2). Both this sequence
and the corresponding amino acid sequence shown in FIG. 1 have been
reported in the GenBank database under accession number P10153.
FIG. 3: Urine cationic protein profiling with SELDI-MS. Urine protein
profiles between 1,000 and 20,000 were displayed according to mass
to charge ratio (m/z). The peaks of interest at 8700 Da (dashed
arrow) and 17400 Da (solid arrow) were prevalent in ovarian benign
tumor and cancer patients, but to a much lesser extent in normal
healthy women.
FIG. 4. Urine biomarker protein purification and mass spectrometry
detection. The identical peak pattern of the peak at 8700 Da (dashed
arrow) and 17400 Da (solid arrow) were present in a cancer case
before (upper panel) and after HiTrap.TM. CM-FF ion exchange column
purification (low panel).
FIG. 5: A. Elevated total EDN protein in sera of ovarian cancer
and benign tumor patients quantified by ELISA. The log-scale box-plot
showed that EDN in urine from benign (n=29, mean=1.26) and cancer
(n=55, mean=1.63) patients was significantly (p<0.001) greater
than that of normal controls (n=30, mean=0.66) by ANOVA. B. The
log-transformed urine and serum EDN concentration from benign and
cancer patients was subjected to Pearson Correlation Coefficient
analysis (n=73). C. The log-transformed blood eosinophil count (%)
and serum EDN (ng per mg protein) concentration from the benign
and cancer patients (n=32) was subjected to Pearson Correlation
Coefficient analysis. D. The CA125 level (Unit per ml) and urine
EDN (ng per mg protein) were log-transformed and subjected to Correlation
Coefficient analysis (n=33).
FIG. 6. Differential glycosylation patterns of EDN dimer in urine
from ovarian benign and malignant cancer patients. A. Summarized
relative protein intensity (the control benign sample as 100%) of
high mobility, minimally glycosylated (solid boxes) and high mobility,
hyperglycosylated forms of EDN (open boxes) from urine of benign
and cancer patients. B. Summarized relative glycosylation intensities
of the minimally glycosylated (solid boxes) and the hyperglycosylated
forms of EDN (open boxes) in urine of benign and cancer patients.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based upon the discovery that one particular
RNase enzyme is selectively elevated in patients with ovarian cancer.
This enzyme is structurally distinct from other RNases that have
been associated with cancer (see, e.g., U.S. Pat. No. 5,866,119)
and is not usually elevated in patients that do not have neoplastic
disease. The amino acid sequence of the protein is shown in FIG.
1 and contains a segment near its C terminal end which is of particular
value in distinguishing it from other members of the RNase family.
The segment is 15 residues in length and reads: RDPPQYPVVPVHLDR
(SEQ ID NO:3). Peptides having this sequence may be synthesized
and used in the generation of antibodies that bind specifically
to EDN.
For the purpose of the present invention, antibodies that bind
specifically are defined as those that have at least a 100 fold
greater affinity for EDN than for other distinct RNases that have
been described in the art. The process for producing such antibodies
may involve either injecting the peptide into an appropriate animal
or injecting peptides that have been conjugated to a protein that
increases the immune response. Methods for making and detecting
antibodies are well known to those of skill in the art as evidenced
by standard reference works such as: Harlow, et al., Antibodies,
Laboratory Manual Cold Spring Harbor Laboratory, NY (1988); Kennett,
et al., Monoclonal Antibodies and Hybridomas: A New Dimension in
Biological Analyses (1980); and Campbell, "Monoclonal Antibody
Technology," in Laboratory Techniques in Biochemistry and Molecular
Biology (1984).
Although the use of polyclonal antibodies is compatible with the
present invention, monoclonal antibodies are generally expected
to give greater specificity. These may be prepared using hybridoma
technology well known in the art (Kohler, et al., Nature 256:495
(1975)). In general, this technology involves immunizing an animal,
usually a mouse, with antigen, e.g., the 15 amino acid peptide described
above. The splenocytes of the immunized animals are extracted and
fused with suitable myeloma cells, e.g., SP.sub.2O cells. After
fusion, the resulting hybridoma cells are selectively maintained
in HAT medium and then cloned by limiting dilution (Wands, et al.,
Gastroenterology 80:225-232 (1981)). The cells obtained through
such selection are then assayed to identify clones which secrete
antibodies capable of specifically binding to EDN.
The antibodies of the present invention may be used to detect the
presence of EDN in any of a variety of immunoassays. For example,
the antibodies may be used in radiommunoassays or immunometric assays,
also known as "two-site" or "sandwich" assays
(see Chard, "Introduction to Radioimmune Assay and Related
Techniques," in Laboratory Techniques in Biochemistry and Molecular
Biology, North Holland Publishing Co., NY (1978)). In a typical
immunometric assay, a quantity of unlabeled antibody is bound to
a solid support that is insoluble in the fluid being tested, e.g.,
blood, plasma, urine, etc. After the initial binding of antigen
to immobilized antibody, a quantity of detectably labeled second
antibody (which, may or may not be the same as the first) is added
to permit the detection and/or quantitation of bound antigen (see,
e.g., Radioimmune Assay Methods, Kirkham, et al., ed. pp. 199-206
E&S Livingston, Edinburgh (1970)). Many variations of these
types of assays are known in the art and may be employed for the
detection of EDN.
In addition to the development of new antibodies and immunoassays
for EDN, assays that have already been described in the art are
suitable for use in the present method. Thus, the radioimmunoassay
described in U.S. Pat. No. 5,928,883 may be used, as may the ELISA
kit and EDN antibodies that are commercially available (Medical
and Biological Laboratories Co., Ltd., Watertown, Mass.).
Although immunoassays are generally preferred, any other procedure
that allows for the quantitation of EDN may also be employed. This
includes immunoblotting assays, HPLC assays or the SELDI mass spectrographic
procedure described more fully in the Examples section. In some
instances, particularly where tissues are removed from a patient
and examined, EDN levels may be determined based upon the amount
of EDN mRNA present. This can be accomplished, for example, using
the polymerase chain reaction in the presence of primers based upon
the sequence shown in FIG. 2. Preferably, at least one of these
primers includes the portion of the EDN sequence coding for the
peptide of SEQ ID NO:3.The most important characteristic of all
of the assays is that they be specific for the detection of EDN.
The selection of an appropriate control population whose samples
will be compared with those derived from a test subject is routine
for one of ordinary skill in the art of clinical assays. Controls
may either be derived from the general population or selected from
individuals believed to be disease free. Although not absolutely
necessary, it is generally desirable to match the characteristics
of controls and test subjects as closely as is practical. For example,
it would generally be desirable for the control population to be
of about the same age as the patient being tested. It should be
recognized that once an EDN level has been established for the control
population, it is not necessary to retest controls in each individual
assay.
At a minimum, the conclusion that a subject is at increased risk
of having or developing ovarian cancer requires that their levels
of EDN be higher than those in the control samples to a degree that
is statistically significant using standard scientific criteria.
Based upon the results obtained using SELDI mass spectrometry, it
is expected that the EDN level in patients with ovarian cancer will
be at least five times higher than in patients that are free from
ovarian disease. However, depending upon the particular assay used,
different cutoff points for concluding that a patient is at risk
may be used. For example, patients evidencing at least a 50% increase
in levels relative to controls in an ELISA assay might be chosen
for additional analysis and monitoring. Similarly, patients evidencing
a twofold, fourfold or tenfold increase in EDN might be chosen.
Although assays of EDN have been established as being useful in
the diagnosis of ovarian cancer, it is expected that these assays
will also find use in helping to diagnose other types of cancer.
Thus, for example, the assays may be used to screen patients for
prostate cancer, breast cancer, or lung cancer. In each case, the
same types of assays described above may be employed. In addition,
the EDN assays may be combined with other assays that suggest the
presence of cancer. For example, in the case of prostate cancer,
EDN assays may be used in conjunction with assays of PSA. Similarly,
subjects being tested for ovarian cancer may be tested both for
EDN levels as well as for levels of CA 125. By combining several
diagnostic tools, a more complete assessment of a subject can be
made.
EXAMPLES
In the present Example, mass spectrometry-based protein chip profiling
on urine samples collected from pre-operative ovarian cancer patients
and age-matched healthy controls is used to identify and characterize
candidate biomarkers associated with ovarian cancers.
A. Materials and Methods:
Urine Sample Collection and Processing
All patient-related biologic specimens were collected and archived
under protocols approved by the Human Subjects Committees of the
Partners HealthCare System, Boston, Mass. Urine samples were collected
pre-operatively from the women requiring surgery for a "pelvic
mass," at Brigham and Women's Hospital (BWH) and Massachusetts
General Hospital (MGH). We randomly collected urine specimens from
84 post-menopausal women before surgery; 55 of them proved to have
epithelial ovarian cancer, 29 had benign gynecologic tumor. A total
of 88 age-matched urine specimens were also available from normal
healthy women selected from the general population from Massachusetts
General Hospital (MGH). The fresh collected urine in sterile tubes
was processed within 8 hours and stored in aliquots at -80.degree.
C.
Mass Spectrometry Profiling of Urinary Proteins
Upon thawing, urine samples were thoroughly mixed and then centrifuged
at 12,000 rpm at 4.degree. C. for 5 min. The protein chip WCX2 (weak
cation exchange, Ciphergen Biosystem, Fremont, Calif.) was activated
by pretreatment with 10 mM of HCl for 5 min. The chip was then washed
twice with 200 .mu.l binding buffer (100 mM ammonium acetate, pH.
6.5) for each spot on the bio-processor. Fifty micro-liters of urine
samples were directly applied on the spot surface and the chip was
inserted into a bioprocessor for incubation on the mini-shaker (IKA-Work
Inc. Wilmington, N.C.) at 600 rpm, at room temperature for 1 hour.
After two washes with 350 .mu.l of the binding buffer, the air-dried
arrays were treated with saturated 3,5-dimethoxy-4-hydroxyinnamic
acid (sinapinic acid) in 0.5% TFA (trifluoroacetic acid) and 50%
acetonitrile before being analyzed by surface enhanced laser desorption/ionization
time-of-flight mass spectrometry (SELDI-TOF-MS) (Protein Biology
System II, Ciphergen Biosystems, Fremont, Calif.). The setting of
mass resolution, accuracy, calibration, and shooting has been previously
described (Ye, et al., Clin Cancer Res. 9:2904-2911 (2003)).
Protein Purification and Identification
A 3 ml urine sample from a patient with documented ovarian cancer
and having the protein pattern of interest (higher intensity at
peak of 8.7 and 17.4 kDa on SELDI-MS profile) was filtered through
a 0.45 .mu.m membrane and mixed with an equal volume of PBS buffer
containing 1.0% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
(CHAPS), and 8 M urea. The sample was then applied to a Sephadex
G-25 column for desalting and removal of the insoluble fraction.
A 1.0 ml HiTrap.TM. CM-FF ion exchange column (Amersham Pharmacia
Biotech AB, Uppsala, Sweden) was utilized for purification according
to the provided protocol. Elution buffers containing 100 mM ammonium
acetate (pH 6.0) and a gradient concentration of sodium chloride
(0-1.0 M) were used. The eluted fractions of interest were subjected
to SELDI-MS to confirm the presence of the desired protein peak
and then separated by 10-20% gradient SDS-PAGE. The separated proteins
were visualized by silver staining. The band of interest was excised
from the gel and subjected to in-gel digestion with trypsin (Shevchenko,
et al., Anal Chem, 68: 850-858, (1996)). The resultant polypeptides
were further separated by liquid chromatography with online sequence
analysis by tandem mass spectrometry (LC-MS/MS) (Mann, et al., Ann.
Rev. Biochem. 70:437-473 (2001); Peng, et al., J. Mass Spectrom.
36:1083-1091 (2001)). The fragmentation ladders (the b and y ion
series) from the lowest mass to the highest mass were used for the
identification of the amino acid residues of the peptides and the
protein identity was searched using a protein database.
Immunoassays and ELISA Quantification
Western blotting: A total of 6 .mu.g of urinary protein prepared
by acetone precipitation from normal and cancer patients was subjected
to 12% SDS-PAGE separation. Proteins were transferred to a PVDF
membrane and then 5% (w/v) of fat-free milk in TBST (10 mM Tris.HCl/100
mM NaCl/0.1% (v/v) Tween-20, pH 7.5) was used for blocking overnight
at 4.degree. C. A polyclonal antibody against human EDN was used
for the primary reaction at a 1:300 dilution in TBST with 5% (w/v)
fat-free milk for 2 hours. The membrane was then washed three times
with Tris Buffered Saline-Tween-20 (TBST) for 15 min per wash. The
secondary antibody was an anti-rabbit IgG coupled to horseradish
peroxidase used according to a protocol for enhanced chemiluminescent
detection (ECL, Pierce).
Immuno-precipitation: A total of 400 .mu.g of urine protein prepared
from acetone precipitation was desalted using a Sephadex G-25 column,
and incubated with 5 .mu.l monoclonal anti-human EDN antibody (MBL
International Inc, Japan) in the washing buffer (20 mM Tris-HCl,
pH. 7.6, 150 mM NaCl, 1 mM MgCl.sub.2, 0.5% NP-40 and 10% glycerol)
for 2 hours at 4.degree. C. Twenty microliters Protein G (Pierce
Biotechnology Inc.) was added for antigen-antibody binding at 4.degree.
C. for overnight. The incubation mixture was centrifuged at 14000
rpm, 4.degree. C. for 5 min. Precipitate pellets were washed three
times with the above washing buffer by centrifugation. The immune-precipitated
protein pellets were dissolved in 20 .mu.l of protein sample buffer
containing 187 mM Tris-HCl, pH 6.8, 30% glycerol, 15% .beta.-mercaptoethanol
and 9% SDS. After boiling for 5 min, the proteins were separated
by 15% SDS-PAGE.
Enzyme-Linked Immunosorbent Assay (ELISA): The urinary EDN protein
concentration was quantified by specific antibody based ELISA, according
to the provided protocol (MBL International Inc. Japan). Urine samples
were thawed and centrifuged at 5000 rpm at 4.degree. C. and transferred
to new tubes. The diluted 150 .mu.l urine samples (1:50) and EDN
standards (0, 0.6, 1.2, 2.4, 4.8, 10, 20, 40 ng/ml) were added into
96-well polyvinyl plates. Each sample (100 .mu.l) was then transferred
to the antibody-coated micro-well plates simultaneously using a
multi-channel pipette followed by a 1-hour incubation at room temperature.
After 4 washes with washing buffer, 100 .mu.l of peroxidase conjugated
anti-human EDN solution were added and followed by a 1 hour incubation.
After another 4 washes, the substrates (mixture of o-phenylenediamine-HCl
and hydrogen peroxide) were added to start the color reaction for
10 min at room temperature. The reaction was stopped by the addition
of 2N sulfuric acid (H.sub.2SO.sub.4). Plates were read at 492 nm
and concentrations of urine EDN were calculated from each standard
curve. The serum EDN and urine eosinophil cationic protein (ECP)
concentrations were measured by using commercial ELISA kits (MBL
International Inc. Japan) according to the provided protocols.
Protein Glycosylation and Dimerization Assay
N-Glycanase.TM. (Peptide-N-Glycosidase F) and O-Glycanase.TM. (Prozyme
Inco. San Leandro, Calif.) were applied to the urine protein sample
for pretreatment according to the provided protocols, which include
the addition of reaction buffer with 0.1% SDS and 50 mM .beta.-mercaptoethanol,
heating at 100.degree. C. for 5 min, 0.75% of NP-40, and incubation
with N-glycanase overnight at 37.degree. C. After glycanase pretreatment,
the protein sample was subjected to SDS-PAGE separation and western
blotting to detect the EDN protein. The total urine protein or the
immune precipitated proteins were separated on SDS-PAGE and followed
by glyco-protein staining with Pro-Q.RTM. Emerald 300 (Molecular
Probes, Eugene, Oreg.). The identical gels for western blot and
for glycoprotein staining were used for identification of specific
glyco-EDN protein bands on the staining gels. The non-specific monoclonal
antibody of mouse anti-human haptoglobin (Sigma-Aldrich) antibody
was applied as the negative control. A concentrated benign urine
protein sample was used as an internal control to normalize the
protein intensity of the individual blots. The western blots and
stained gels were scanned using an Imaging Densitometer (Model GS-700)
and analyzed with the Quantity One software (Bio-Rad). The relative
intensities of EDN protein on western blots and glyco-staining gels
were analyzed.
Urinary Creatinine and Protein Quantification
Concentrated urine protein samples were prepared by acetone precipitation.
The total urine creatinine concentration was measured by using the
Creatinine Companion assay kit (Exocell, Inco. Philadelphia, Pa.)
according to the protocol provided. The total urine protein concentration
was measured by the BCA assay using the kit provided from Pierce
Biotechnology, Inc. (Rockford, Ill.).
Statistical Analysis
The urine cationic protein profiling data and the intensity of
the protein peaks of interest from benign ovarian tumor patients,
ovarian cancer patients, and normal controls were compared using
Student's t-test. The EDN concentration of the urine and serum quantified
by ELISA was normalized by total protein or creatinine concentration.
The measurements were transformed to the logarithmic scale and t-tests
were used to compare the distributions in ovarian cancers, ovarian
benign tumors and age-matched normal controls. The log-transformed
urine EDN concentrations from the non-mucinous cancer patients and
normal controls were used to generate a receiver operating characteristic
(ROC) curve and to assess sensitivity at a fixed specificity level.
To validate the sensitivity estimate, "leave-one-out"
cross-validation was used, whereby one observation is iteratively
omitted to obtain urine EDN cut-point, which is then used to classify
the omitted observation. To evaluate the relationship between EDN
concentration and other relevant serum measurements, correlation
coefficients were estimated and linear models were fit comparing:
1) log-transformed urine EDN concentration against log-transformed
serum EDN; 2) log-transformed serum EDN concentrations against blood
eosinophil; and 3) log-transformed CA125 measurements against log-transformed
urine EDN.
B. Results
Urinary Protein Mass-Spectrometry Profiling and Liquid Chromatography
Purification
To study the relevant cationic proteins in ovarian cancer urine
specimens, we utilized the SELDI-TOF-MS to generate protein profiles
using the weak-cationic exchange (WCX) surface chip. Two cationic
protein peaks at 8730 and 17400 m/z were identified which had a
significantly (p<0.001) higher peak intensity in benign ovarian
tumor patients (7.72, n=29) and cancer patients (13.8, n=42) compared
to normal controls (0.55, n=55) in the preliminary SELDI screening
(FIG. 3). The candidate protein identified from cancer patients
was purified using the cationic ion exchange chromatography. The
eluted fractions were applied on SELDI-MS protein chip to confirm
presence of the protein mass. Most of the urine proteins were removed
from column purification steps and not detected by mass spectrometry.
The enriched candidate urine cationic proteins were shown to have
the identical mass spectrometry pattern as in the original screening
profiles (FIG. 4). The purified cationic protein fraction was concentrated
and separated on an 8-16% gradient SDS-gel and viewed by the silver
staining. A lower band on the gel was observed with a molecular
weight of .about.17 kDa. In addition, an upper band was present
indicating the presence of a second form of higher molecular weight
(.about.35 kDa).
Eosinophil-Derived Neurotoxin (EDN): Identification, Characterization
and Quantification
The purified urine cationic protein was excised from the gel and
subjected to trypsin digestion. The resultant peptides were further
then subjected to LC-MS/MS for sequence analysis. Using this procedure,
the cationic protein was identified as eosinophil-derived neurotoxin
(EDN, also known as RNase 2) with the unique C-terminal peptide
sequence, RDPPQYPVVPVHLDR (SEQ ID NO:3). The elevation of EDN in
urine samples was confirmed by western blot and showed that EDN
protein was elevated in the urine from cancer patients, with at
least three different forms in the molecular weight range of 17-20
kDa, due primarily to glycosylation differences. To quantify the
urine EDN, ELISAs were performed in 55 patients with ovarian cancer,
29 with benign ovarian tumor, and 88 age-matched healthy controls
using a commercially available assay (MBL International Inc. Japan).
EDN protein levels were normalized either by the total amount of
urine protein or by urine creatinine concentration. The log-transformed
mean of EDN concentration (ng per mg protein) in the urine of patients
with benign ovarian tumor and cancer patients were 92.0 and 105.9
respectively, which was significantly different from the normal
controls which had a mean value of 24.4 (p<0.001). A ROC curve
illustrated the maximum specificity (94%) and sensitivity (72.2%)
of urine EDN to separate the cancer cases from the normal controls.
However, eosinophil cationic protein (ECP), another eosinophil ribonuclease
with 67% amino acid sequence homology to EDN, was not present in
the urine of cancer patient or in normal controls either by SELDI
mass spectrometry profiling or by ELISA quantification.
The mean value of urine EDN protein and 95% confidence interval
(CI) of patients with benign ovarian tumor (92, 61.0-138.6) and
ovarian cancer (105.9, 85.5-131.1) were significantly different
(p<0.001) from that in normal controls (24.2, 20.7-28.4) (Table
1). The total urine EDN protein concentration in benign ovarian
tumor was not significantly different from the ovarian cancers.
In addition, among all subtypes of ovarian cancers, the mucinous
subtype was shown to have the lowest levels of urine EDN. Using
94% specificity as cutoff (75 ng/mg protein), urine EDN levels in
the early stages of ovarian cancer (72.2% sensitivity) was comparable
to that in the late stages of ovarian cancers (69% sensitivity).
The non-mucinous ovarian cancer patients can be detected with 82.3%
sensitivity (Table 1).
Coordinate Elevated Blood Eosinophil, Serum EDN, CA125 and Urine
EDN in Ovarian Cancer Patients
The elevated EDN level in the urine of ovarian cancer patients
may be associated with an elevated serum EDN level and blood eosinophil
count, as opposed to EDN synthesis from ovarian cancer cells directly.
To test this hypothesis, serum EDN levels were measured with the
above commercial available ELISA kit using a standard protocol.
The log transformed mean values of serum EDN (ng/mg protein) from
the benign ovarian tumor patients and ovarian cancer patients were
1.26 and 1.63, respectively, and significantly (p<0.001) higher
than that in the controls (mean value=0.66 ng/mg protein). The increased
urine EDN has a strong correlation tendency with the elevated serum
EDN (n=73, correlation coefficient: r=0.298, p=0.0105). The possible
correlation of elevated serum EDN with the increased blood eosinophil
level was further tested. The combined blood eosinophil count percentile
data of the benign ovarian tumor and cancer patients collected from
our clinical database was plotted with their serum EDN concentration
(ng/mg protein) in log-transformed scale. It clearly indicated that
the increased incidence of high EDN level in serum was strongly
correlated with blood eosinophil count (n=32, correlation coefficient:
r=0.485, p=0.0049). Interestingly, the increased serum CA125 level
was significantly correlated with the increased urine EDN in the
benign and ovarian cancer patients (n=35, correlation coefficient:
r=0.458, p=0.0074).
Hyperglycosylated EDN in Ovarian Cancer
The polypeptide sequence of EDN contains 5 potential sites for
N-linked glycosylation. To evaluate whether the higher molecular
weight form of EDN found in urine from benign ovarian tumor and
cancer patients resulted from glycosylation, N-glycanase or O-glycanase
was used to remove all potential glycosyl groups from the EDN polypeptide.
After pretreatment with 0.1% SDS, 50 mM .beta.-mercaptoethanol,
0.75% NP-40, with either N-glycanase, or O-glycanase (ProZyme, Inc.
San Leandro, Calif.) incubation at 37.degree. C. overnight, western
blots showed that the high molecular weight, lower mobility, hyperglycosylated
form of EDN disappeared and transformed to the lower molecular weight,
high mobility form. The lower molecular weight, high mobility form
of EDN was also slightly shifted to an even smaller size by glycanase
treatment. This indicated that the mature monomer EDN was likewise
slightly, or minimally glycosylated. Pretreatment control conditions
(without glycanase) caused partial transformation of the glycosylated
form of EDN. To further differentiate EDN in ovarian cancer and
benign tumor patients, immune precipitation was used to purify EDN
from the pooled urine specimen of the benign tumor (n=9) and cancer
patients (n=12). A western blot showed that the immune precipitated
hyperglycosylated form of EDN (upper band in gels) has a greater
intensity in the cancer patients than that of the benign tumor patients.
The minimally glycosylated form of EDN (lower band in gels) however,
was shown to have similar intensities in the cancer and benign patients.
The identical gel was also used for glycosylation staining. Interestingly,
hyperglycosylated EDN was shown with much greater intensity in the
cancer patients than that in benign tumor patients. There was no
detectable difference in glycosylation intensity of the lower molecular
weight form of EDN. The increased hyperglycosylated EDN protein
intensity in urine of ovarian cancer patients was shown on the western
blots, but much less difference for the lower molecular weight,
minimally glycosylated form of EDN. The relative abundance of the
minimally and the hyperglycosylated forms EDN of 9 benign tumor
and 12 cancer patients is summarized in FIG. 6. The hyperglycosylated
EDN was about 2-fold higher (p<0.001) in cancer urine after normalized
with the total urine protein. This finding is consistent with our
SELDI-mass profiling data that peak intensity of 17.4 kDa was 2-fold
higher in urine of cancer patients than that in benign tumor likely
due to enhanced chip surface binding of glycosylated form of EDN.
However, the relative glycosylation-intensity of the hyperglycosylated
EDN was .about.6 times higher (p<0.001) in ovarian cancer urine
than that from the benign patients. There was less detectable difference
of the glycosylation intensity of minimally glycosylated EDN between
the benign and malignant cancer patients.
C. Discussion
Eosinophils are rare granulocytes that are normally associated
with allergic diseases or response to inflammation due to various
parasitic infections and immune reactions. However, many types of
human cancers are associated with extensive eosinophilia within
the tumor tissue (Schwartz, R., N. Engl. J. Med. 348:1199-1200 (2003);
Samoszuk, M., Histol. Histopathol. 12:807-812 (1997)), which include
oral squamous cell carcinomas (Dorta, et al., Histopathology 41:152-157
(2002)), skin (Suster, S. Semin. Diagn. Pathol. 16:162-177 (1999)),
breast (Pastrnak, et al., Neoplasma 31:323-326 (1984); Ali, et al,
Am. J. Pathol. 157:313-321 (2000)), lung (Kodama, et al., Cancer
54:2313-2317 (1984)), and colorectal (Fernandez-Acenero, et al.
Cancer 88:1544-1548 (2000)). The present Example demonstrates a
significant (p<0.001) increase of urine EDN in ovarian cancer
patients and correlation with the increased levels of blood eosinophil
and serum EDN. It may be that the elevated urine EDN is not derived
from ovarian cancer cells, but rather from the indirect pathological
and immune reactions, which may include the eosinophil activation
and degranulation. By using autofluorescence and immunohistochemistry,
Samoszuk's group has shown the presence and degranulation of eosinophils
in breast and ovarian cancer tissues (Samoszuk, et al., Clin. Cancer
Res. 2:1867-1871 (1996); Samoszuk, et al., Am. J. Pathol. 148:701-706
(1996)) and in endometriosis (Blumenthal, et al. Am. J. Pathol.
156:1581-1588 (2000)). This observation may be relevant to our finding
that EDN is especially apparent in women with endometrioid types
of ovarian cancer and endometriomas (12 of 14 patients, Table 1).
EDN is a pyrimidine specific nuclease of the RNase A gene superfamily,
also known as RNase 2, and it possesses several characterized biological
activities. EDN is a major component of human eosinophilic leukocytes.
Increased serum ribonuclease activity has been reported in women
with ovarian carcinoma (Schleich, et al., J. Cancer Res. Clin. Oncol.
113:603-607 (1987)). But it was not clear if the high level of RNase
activity is due to the elevation of EDN in serum because the EDN
has >1000-fold RNase activity than other members of RNase proteins.
Intact eosinophils as well as the "footprints" of cell
undergoing degranulation were found in blood vessel adjacent to
tumor in ovarian cancer (Samoszuk, M. Histol. Histopathol. 12:807-812
(1997)). Interestingly, the post-translational modification with
N-terminal extension of amino acid residues -4 to -1 of signal peptide
EDN has inhibitory activity of oocyte maturation (Sakakibara, et
al., Chem. Pharm. Bull. (Tokyo) 39:146-149 (1991)) and is predominantly
present in pregnant women (Sakakibara, et al., J. Biochem (Tokyo)
111:325-330 (1992)). These findings together with our data of elevation
of urine EDN in ovarian cancer patients strongly suggest that the
activated eosinophil and EDN modification might cause abnormal ovarian
functions.
We have further shown that the urine from ovarian cancer patients
has about 6 times more hyperglycosylated EDN than urine from patients
with benign ovarian tumor patients. Recent novel discoveries have
shown that cancer malignancy is linked with protein glycosylation
via the inhibition of apoptosis signaling pathway (Hakomori, et
al., Proc. Nat'l Acad. Sci. USA 99:10231-10233 (2002); Kakugawa,
et al., Proc. Nat'l Acad. Sci. USA 99:10718-10723 (2002); Alper,
J., Science 301:159-160 (2003)). We suggest that post-translational
modifications including glycosylation of EDN might play essential
roles in ovarian cancer development. The complexity of EDN glycosylation
may include the glycan types, structure and glycan binding sites.
With a clear understanding of the biochemical properties of glycosylated
EDN from the ovarian cancer patients, we could generate more specific
antibodies to improve specificity and sensitivity for the urine
test and to have better understanding of eosinophil related pathways
in ovarian cancers.
TABLE-US-00001 TABLE 1 Comparison of clinical characteristics and
urine eosinophil-derived neurotoxin (EDN) in ovarian benign, cancer
patients and the age-matched normal postmenopausal controls. Eosinophil-Derived
Neurotoxin (ng/mg protein) Characteristic Patients Mean 95% CI Range
Positive (%) p-value Normal Total 88 24.2 20.7-28.4 6.4-112.4 5
Benign Serous cystadenoma 6 89.6 25.2-319.0 22.7-350.5 40 Endometrosis
3 129.0 27.6-602.4 79.3-259.4 75 Mucinous 2 94.6 0.2-58227.9 57.1-156.8
50 Other 18 87.4 48.6-157.1 7.9-465.2 72 Total 29 92.0 61.0-138.6
7.9-465.3 66 <0.001 Ovarian Cancer Histotype Serous 40 113.1
90.3-141.7 26.2-377.1 72 Endometroid 3 96.7 20.6-454.6 57.2-192.5
67 Mucinous 5 52.5 24.3-113.4 28.5-147.4 20 Other 8 132.5 55.0-319.2
21.9-475.6 73 Non-mucinous 51 114.9 93.0-141.8 21.9-475.6 74 <0.001
Stage (non-mucinous) I-II 22 95.1 68.2-132.7 28.5-245.9 72.2 <0.001
III-IV 31 112.9 84.2-151.6 21.9-475.6 69 <0.001 Total 55 107.1
87.2-131.6 21.9-475.6 66 <0.001
All references cited herein are fully incorporated by reference.
Having now fully described the invention, it will be understood
by those of skill in the art that the invention may be performed
within a wide and equivalent range of conditions, parameters and
the like, without affecting the spirit or scope of the invention
or any embodiment thereof. |