Note: Descriptions are shown in the official language in which they were submitted.
OVARIAN CANCER BIOMARKER AND METHODS OF USING SAME
FIELD OF INVENTION
[0001] This invention relates to a novel ovarian cancer biomarker and to
related methods,
uses, agents, and kits. More specifically, the invention relates to methods of
diagnosing,
prognosing, and treating ovarian cancer using the biomarker. The invention
also relates to
methods of assessing the severity of ovarian cancer and monitoring responses
to treatment for
ovarian cancer using the biomarker.
BACKGROUND
[0002] Ovarian cancer remains a major health concern worldwide,
accounting for 6% of
all cancer deaths [1]. It is the second most common gynecological neoplasm,
with over 2,800
new cases diagnosed in Canada in 2016 alone, more than 1,800 deaths in the
same year [2], and
3,100 new cases diagnosed in Canada in 2020 [34]. In Canada, the 5-year net
survival for
ovarian cancer is approximately 44%; the survival rate has only modestly
increased by 2 to 4%
since 1995.
[0003] Ovarian cancer is a general term for a group of neoplasms
originating from the
ovary, the majority (about 90%) of which are classified as epithelial
carcinomas. Epithelial
ovarian cancers (E0Cs) comprise several morphologically distinct groups:
serous, mucinous,
endometrioid, clear cell, transitional cell, squamous cell, and mixed
epithelial neoplasms [3].
[0004] High-grade serous ovarian cancer (HGSOC) is an aggressive subtype
that
accounts for approximately 80% of all ovarian-cancer-related deaths.
Worldwide, HGSOC is the
eighth most frequent cause of cancer-related deaths in women [4]. Most HGSOC
patients
present with advanced disease and undergo surgical debulking followed by
chemotherapy (e.g.,
combinations of platinum drugs and paclitaxel) [5]. Their cancers commonly
relapse within two
years and develop broad chemoresistance, leading to a very poor prognosis [6].
[0005] Over the past decade, there has been relatively little improvement
in ovarian
cancer survival rates. The majority of ovarian cancer cases remain
asymptomatic in the early
stage and present at an advanced stage, at which point the disease is rarely
curable by existing
standards of care. As a consequence, ovarian cancer shows the highest
mortality rate among
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gynecologic cancers, with only a 29% 5-year survival rate for advanced ovarian
cancer.
Importantly, disease outcome is significantly higher (5-year survival rates
over 90%) with early
diagnosis in stages I and II [7-9].
[0006] Accordingly, there exists a need for additional options, including
biomarkers,
for early detection of primary ovarian cancer. Such biomarkers, including
plasma biomarkers,
can be useful in periodic screening of asymptomatic women for ovarian cancer
and also as
diagnostic tools for detecting ovarian cancer in women with a broad range of
unspecific
symptoms of ovarian cancer.
SUMMARY
[0007] As described in further detail herein, the inventor has
surprisingly determined that
the Arresten polypeptide or a portion thereof can be used as a biomarker for
cancer, particularly
ovarian cancer. These discoveries have broad implications in the diagnosis,
prognosis,
monitoring, and treatment of cancer, including ovarian cancers, and especially
type II ovarian
cancers, which typically could be diagnosed not earlier than stage II.
[0008] Thus, in one aspect, the present application provides a method for
determining
whether a subject has ovarian cancer, by assaying a diagnostic sample of the
subject for Arresten
expression, where detection of Arresten expression elevated above normal is
diagnostic of
cancers, particularly ovarian cancer, in the subject. Also provided is a
method of screening a
general population for ovarian cancer, by testing a plurality of asymptomatic
subjects in
accordance with the above method.
[0009] Additionally, the present application provides a method for
treating ovarian
cancer in a subject, by analyzing a diagnostic sample of the subject for
Arresten expression, and
providing a therapy to the subject when the Arresten expression is elevated
above normal. By
way of example, the therapy can comprise at least one of: surgical debulking,
chemotherapy, and
radiation therapy. In certain embodiments, the ovarian cancer is high-grade
serous ovarian
cancer of stage I-TV (e.g., stage I-IIIb).
[0010] The present application also provides a method for assessing the
severity of the
disease in a patient who has been diagnosed with ovarian cancer. The method
includes assaying
a biological sample of the patient for Arresten expression prior to treatment
and then either:
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(a) determining a positive or favorable prognosis when Arresten expression in
the biological
sample is normal, or (b) determining a negative or poor prognosis when
Arresten expression in
the biological sample is elevated above normal.
[0011] The present application further provides a method for assessing
the efficacy of
therapy to treat ovarian cancer in a subject who has undergone or is
undergoing treatment for
ovarian cancer. In accordance with this method, the efficacy of the therapy
can be assessed by
assaying a first diagnostic sample of the subject for Arresten expression
after therapy has
commenced, obtaining a first level of Arresten expression in the first
diagnostic sample, and then
comparing the first level with a second level of Arresten expression in a
second diagnostic
sample of the same subject, where the second diagnostic sample was assayed and
the second
level was obtained prior to the therapy. A significant decrease of the first
detected level, relative
to the second level, can indicate that the subject is responding to the
therapy to treat ovarian
cancer; a minor or no decrease of the first detected level, relative to the
second level, can indicate
that the subject is not responding to the therapy to treat ovarian cancer. In
some embodiments,
the first level is below a concentration selected from 80-100 pg/ml, and the
second level is above
the selected concentration.
[0012] The present application also provides a method for assessing the
prognosis of a
subject who has ovarian cancer, by assaying a diagnostic sample of the subject
for Arresten
expression. In accordance with this method, the subject's prognosis improves
with a decrease in
Arresten expression in the diagnostic sample, and the subject's prognosis
worsens with an
increase in Arresten expression in the diagnostic sample.
[0013] Additionally, the present application provides a method for
diagnosing ovarian
cancer in a human female subject or for screening a human female subject for
ovarian cancer
when the subject is asymptomatic of ovarian cancer. The method includes: (a)
isolating Arresten
that is present in a diagnostic sample of the subject by immobilizing the
Arresten on a solid
surface; (b) forming a complex of the immobilized Arresten with a primary
antibody specific for
Arresten, where the complex is coupled with an enzyme to form an enzyme
complex;
(c) incubating the enzyme complex with a substrate for the enzyme; (d)
measuring a detectable
signal produced by the enzyme acting on the substrate; and (e) calculating a
level of Arresten in
the diagnostic sample based on the measurement of the detectable signal. In
accordance with
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this method, a level of Arresten elevated above a predetermined cut-off value
is diagnostic of
ovarian cancer in the subject. The enzyme in the enzyme complex can be
conjugated to at least
one of the following: (a) the primary antibody; (b) a secondary antibody that
binds to the primary
antibody; and (c) a protein (e.g., streptavidin or avidin, whether native or
modified by
glycosylation or deglycosylation) that binds to biotin labelling the primary
antibody or the
secondary antibody. The immobilized Arresten can be directly immobilized to
the solid surface
or indirectly immobilized to the solid surface through a capture antibody that
is coated on the
solid surface and specifically binds to Arresten. In some embodiments, the
method further
includes comparing the detectable signal to a calibration data set generated
using a calibrator
(e.g., recombinant human Arresten) at multiple concentrations or levels to
assess amount,
concentration, or level of Arresten in the diagnostic sample. In some
embodiments, the
detectable signal is measured by colorimetry, immunofluorescence,
bioluminescence, or
chemiluminescence.
[0014] In a further aspect, the present application provides a method for
treating ovarian
cancer (e.g., by surgical debulking, chemotherapy, radiation therapy) in a
subject who was
diagnosed with ovarian cancer by a process that includes: (a) obtaining a
serum or plasma
sample from the subject; (b) analyzing the sample for Arresten expression; and
(c) detecting
Arresten expression elevated above normal, thereby diagnosing the subject with
ovarian cancer.
By way of example, the process can have a sensitivity of at least about 60%
and a specificity of
at least about 75%. The process can also be based on a receiver operating
characteristic (ROC)
curve with an area under the curve (AUC) of at least about 0.70.
[0015] In the methods of the present invention described herein, the
diagnostic sample
(e.g., plasma, urine, etc.) can be assayed using an agent reactive with
Arresten. In some
embodiments, the agent is an antibody or an antigen-binding fragment thereof.
The agent can
also be labeled with a detectable marker. Furthermore, the diagnostic sample
can be assayed
using an ELISA, a chemiluminescence assay, an immunohistochemistry assay, and
the like. In
certain embodiments, Arresten expression is considered to be elevated above
normal when the
Arresten expression is above a concentration selected from 80-100 pg/ml.
[0016] Additionally, in the methods of the present invention described
herein, the subject
or patient may be at least one of the following: (a) pre-menopausal; (b)
asymptomatic of ovarian
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cancer; (c) not carrying a mutation of BRCA1 or BRCA2 gene; (d) suffering from
a non-
malignant gynecologic disease, a peritoneal, pleural, or musculoskeletal
inflammatory disorder, a
pelvic inflammatory disease, a liver, renal, or cardiac disease, or an
advanced adenocarcinoma.
In some embodiments, the ovarian cancer is a type II ovarian cancer,
particularly at stage I, II,
Ma, or Mb. By way of example, the ovarian cancer may be undetectable by
existing tests, such
as CA125 test or a transvaginal ultrasound.
[0017] In some embodiments of the present invention, Arresten expression
can be
detected in conjunction with the detection of at least one additional
biomarker, such as CA125 or
HE4. In particular, expression of CA125 can be detected in addition to
Arresten. An exemplary
cut-off value for Arresten expression can be in the range of 80-100 pg/ml; an
exemplary cut-off
value for CA125 can be in the range of 35-100 U/ml.
[0018] In some embodiments of the present invention, the predetermined
cut-off value is
80-100 pg/ml; or the predetermined cut-off value is set so that the method
has: (a) a sensitivity of
at least about 60%; (b) a specificity of at least about 75%; or (c) a
sensitivity of at least about
60% and a specificity of at least about 75%. For example, the predetermined
cut-off value can
be set so that the method has a specificity or sensitivity of about 90% or
greater. The
predetermined cut-off value can be determined based on a receiver operating
characteristic
(ROC) curve with an area under the curve (AUC) of at least about 0.70 or at
least about 0.85.
The predetermined cut-off value can also be based on at least one of the
following: (a) a type of
ovarian cancer and (b) a stage of ovarian cancer. In some embodiments, the
predetermined cut-
off value is specific to stage II ovarian cancer and set so that the method
has a sensitivity of least
about 60%.
[0019] In other aspects, the present application provides kits for
diagnosing, detecting,
and assessing risk of ovarian cancer. For example, in one aspect, the present
application
provides a kit for use in detecting ovarian cancer, including an agent
reactive with Arresten and
at least one reagent suitable for detecting expression of Arresten. The kit
can be adapted or
configured for ELISA-based point-of-care testing. The kit can also include a
second agent
reactive with another biomarker, such as CA125, and at least one second
reagent suitable for
detecting expression of the other biomarker.
[0020] In another aspect, the present application provides a diagnostic
kit for assessing
Date Recue/Date Received 2022-06-16
risk of ovarian cancer in a female subject by measuring a level of Arresten
expression in a
biological sample obtained from the subject. The kit can include: (a) a
capture antibody that is
capable of specifically binding to human Arresten, thereby isolating human
Arresten from the
biological sample; (b) a solid matrix to which the capture antibody will bind;
(c) a detection
antibody that is capable of specifically binding to human Arresten and has a
label for generating
a detectable signal; (d) a recombinant human Arresten standard for
calibration; and (e) at least
one reagent suitable for generating the detectable signal in cooperation with
the label, thereby
detecting a level of human Arresten in the biological sample.
[0021] In some embodiments, the diagnostic kit can also include: (a) a
second capture
antibody that is capable of specifically binding to human CA125, thereby
isolating human
CA125 from the biological sample; (b) a second detection antibody that is
capable of specifically
binding to human CA125, and has a second label for generating a second
detectable signal; (c) a
recombinant human CA125 standard for calibration; and (d) at least one second
reagent suitable
for generating the second detectable signal in cooperation with the second
label, thereby
detecting expression of human CA125. In this context, a predetermined cut-off
value of the
human Arresten can be, for example, 80-100 pg/ml, and a predetermined cut-off
value for the
human CA125 can be, for example, 35-100 U/ml.
[0022] In a further aspect, the present application provides a device for
determining
whether a subject has ovarian cancer, by detecting Arresten expression in a
urine sample of the
subject. By way of example, the device can include a housing, a matrix of
absorbent material,
and an immunoassay strip. In certain embodiments, the housing contains the
matrix and the strip
(e.g., at least partially coated with a labeled anti-Arresten antibody). In
use, a urine stream or
urine sample of the subject can contact the absorbent matrix or pad of the
device, which draws
the liquid by capillary action into the immunoassay strip. Exemplary absorbent
materials for use
in the device of the present invention include nitrocellulose, polysulfones,
polycarboxylic acids,
filter paper, and the like.
[0023] As discussed in further detail below, the present application
identifies Arresten as
a new biomarker for ovarian cancer, with a sensitivity and specificity that
are comparable to the
existing biomarkers (e.g., CA125, HE4). Also described are uses of an antibody
specific to
Arresten for the diagnosis, treatment, or prognosis of ovarian cancer in a
subject.
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[0024] Using the methods, kits, antibodies, and devices of the present
invention, it is
possible to detect ovarian cancer, particularly type II ovarian cancer, at
early stages of the
disease (e.g., stage II). Moreover, it is possible to screen asymptomatic
women periodically for
ovarian cancer, and to detect ovarian cancer in women with a broad range of
unspecific
symptoms of ovarian cancer.
[0025] Additional aspects of the present invention will be apparent in
view of the
description which follows.
BRIEF DESCRIPTION OF THE FIGURES
[0026] Embodiments of the invention will now be described, by way of
example only,
with reference to the accompanying drawings, in which:
[0027] Figure 1 shows plasma concentrations of Arresten (pg/ml) in
hospital controls
versus ovarian cancer patients;
[0028] Figure 2 shows a receiver operating characteristic (ROC) analysis
of Arresten
levels in ovarian cancer cases; and
[0029] Figure 3 shows ROC analyses of Arresten levels in ovarian cancer
cases separated
by stage.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Arresten is a protein segment from the C-terminal of the collagen
IV alpha chain,
released due to collagen IV breakdown during extracellular matrix exposure in
metastasis,
starting at stage II. Prior to the present invention, Arresten had not been
investigated in the
context of ovarian cancer and has never been considered as a potential serum
biomarker for
ovarian cancer. The existing literature did not support use of Arresten as an
ovarian cancer
biomarker. The inventor's results, as disclosed herein, now identify Arresten
as a new biomarker
for diagnosis of ovarian cancer, especially ovarian cancer in stage II and
higher.
[0031] More specifically, the inventor demonstrates herein a significant
increase of
Arresten expression in diagnostic samples from ovarian cancer patients, as
compared with
healthy controls (see, e.g., Figure 1). Accordingly, in one aspect, the
present invention provides
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methods for determining whether a subject has ovarian cancer. The methods can
include
assaying a diagnostic sample of the subject for expression of Arresten, where
detection of
Arresten expression elevated above normal is diagnostic of neoplasia,
particularly ovarian
cancer, in the subject.
[0032] The Arresten biomarker disclosed herein can be used in methods for
the
diagnosis, prognosis, treatment, and monitoring of cancer, particularly
ovarian cancer. In some
embodiments, the methods of the present invention can be used to discriminate
between healthy
subjects and cancer subjects, including subjects with early-stage (e.g., stage
II) disease. The
methods can be based on the early detection, identification, or quantification
of the Arresten
biomarker, which is particularly well-suited to discriminate between healthy
subjects and ovarian
cancer subjects. The cancer subjects can include asymptomatic subjects and/or
those at an early
stage of the disease.
[0033] As a biomarker of ovarian cancer, Arresten can be detected in a
biological
sample, either alone or in combination with additional known biomarkers. By
way of example,
the biomarker can be detected, identified, or quantified by a screening method
using biological or
diagnostic samples from subjects. For instance, the biomarker can be detected
in a blood or
urine test. Known biomarkers of ovarian cancer include, without limitation,
Cancer Antigen 125
(CA125) and Human Epididymis Protein 4 (HE4). Detection of Arresten expression
in
conjunction with detection of CA125 and/or HE4 can be particularly useful in
the early detection
of ovarian cancer and may significantly improve the accuracy of detecting pre-
malignant
changes or early-stage ovarian cancers in asymptomatic women at increased risk
for the
development of ovarian cancer.
[0034] Moreover, as a biomarker of ovarian cancer, Arresten can be
detected and
quantified in a biological sample in conjunction with other diagnostic
techniques. For example,
a test based on Arresten as a biomarker can be used in conjunction with
vaginal examination,
ultrasound, or MRI to diagnose ovarian cancer.
[0035] As noted above, ovarian cancer is asymptomatic in the early stages
and most
patients present with advanced levels of the disease. Cost-effective and non-
invasive methods
that can promote frequent testing may achieve early detection and high
survival rates in ovarian
cancer patients. Accordingly, in some embodiments of the methods described
herein, Arresten
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can be detected through non-invasive tests. For example, subjects can be
screened for Arresten
expression using a simple and inexpensive detection module based on the well-
known enzyme-
linked immunosorbent assay (ELISA). This option can be useful for detecting
Arresten in urine
samples from subjects, including ovarian cancer patients.
[0036] Other objects, features, and advantages of the invention will
become apparent
from the following discussion. It should be understood, however, that the
specific examples and
preferred embodiments of the invention described herein are given by way of
illustration only,
and various changes and modifications within the scope of the invention will
become apparent to
those skilled in the art from the description which follows.
Definitions
[0037] The following definitions are presented as an aid to understand
the invention.
[0038] The term "subject" as used herein refers to a mammal, including,
without
limitation, a cow, dog, human, monkey, mouse, pig, or rat. Preferably, the
subject is a human.
More preferably, the subject is a woman.
[0039] The terms "sample", "biological sample", "diagnostic sample", and
the like, as
used herein, refer to a material known or suspected of expressing or
containing one or more
cancer markers. The diagnostic sample can include any bodily fluids, tissues,
or cells (e.g.,
blood, serum, plasma, urine, saliva, ovary tissues, mammary tissues, etc.).
The sample is
preferably a bodily fluid sample, such as blood, serum, plasma, vaginal
secretions, urine, tears,
saliva, etc.
[0040] As used herein, "blood" or "blood sample" can include a sample of
whole blood,
serum, or plasma, unless a different meaning is specified.
[0041] The terms "cancer" and "neoplasm" refer to a proliferation of
tumour cells in
tissue having the unique trait of loss of normal controls, resulting in
unregulated growth, lack of
differentiation, local tissue invasion, and/or metastasis, and include
malignant tumours that are
either invasive or non-invasive.
[0042] The phrase "primary cancer" refers to a cancer that is at a
location of the body or a
tissue where the particular cancer starts. Primary cancer is the opposite of
metastasis, which
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refers to the migration of cancer cells from the original tumour site to
produce cancer in other
tissues. For example, a cancer originating in the ovary is called a "primary
ovarian cancer". If it
metastasizes and spreads to the liver, the cancer is considered a primary
ovarian cancer
metastatic to the liver.
[0043] The terms "type I" / "subtype I" and "type II" / "subtype II" are
used herein to
refer to cancers, particularly ovarian cancers, which have remarkably
different molecular genetic
features as well as morphologic differences. For example, high-grade serous
carcinoma (type II)
is characterized by very frequent TP53 mutations, but rarely has mutations
that characterize most
type I carcinomas, including KRAS, BRAF, ERBB2, PTE1V, CTNNB1, and PIK3CA. In
general,
type I tumours are genetically more stable than type II tumours and display a
distinctive pattern
of mutations that occur in specific cell types. Type II tumours which show
greater morphologic
and molecular homogeneity are genetically unstable and have a very high
frequency of TP53
mutations. Therefore, it has been suggested that these two different types of
ovarian cancers
develop along different molecular pathways.
[0044] The term "marker" or "biomarker" refers to an indicator which can
be detected in
a sample, and includes predictive, diagnostic, and prognostic indicators and
the like. The
biomarker can be an indicator of a particular disease or disorder (e.g.,
ovarian cancer or other
cancer), having certain molecular, pathological, histological, and/or clinical
features.
[0045] The "presence", "amount", "concentration", or "level" of a marker
associated with
an increased clinical benefit or disadvantage to an individual includes a
detectable level of the
marker in a sample. The presence, amount, or level of a marker can be measured
by methods
known to a person skilled in the art. Furthermore, the presence, amount, or
level of a marker can
be measured prior to treatment, during treatment, after treatment, or a
combination of any of the
foregoing.
[0046] As used herein in connection with Arresten, the term "expression"
includes,
without limitation, the transcription of the Arresten-associated gene into at
least one mRNA
transcript and/or the translation of at least one mRNA transcript into an
Arresten protein.
Accordingly, a diagnostic sample can be assayed for Arresten expression by
assaying for
Arresten, Arresten cDNA, or Arresten mRNA. The appropriate form of Arresten
will be
apparent based on the particular techniques discussed herein.
Date Recue/Date Received 2022-06-16
[0047] The phrase "elevated above normal", as used herein, refers to
expression of
Arresten that is detected at a level significantly greater than the level
expected for the same type
of diagnostic sample taken from a non-diseased subject or patient (i.e., one
who does not have
cancer, such as ovarian cancer) of the same gender and of similar age. As
further used herein,
"significantly greater" refers to a statistically significant difference
between the level of Arresten
expression elevated above normal and the expected (normal) level of Arresten.
Preferably,
Arresten expression that is elevated above normal is expression of Arresten at
a level that is at
least 10% greater than the level of Arresten expression otherwise expected.
Where Arresten
expression is expected to be absent from a particular diagnostic sample taken
from a particular
subject or patient, the normal level of Arresten expression for that subject
or patient is nil.
Where a particular diagnostic sample taken from a particular subject or
patient is expected to
have a low level of constitutive Arresten expression, that low level is the
normal level of
Arresten expression for that subject or patient.
[0048] A "reference sample" or "control sample", as discussed herein, is
a biological
sample provided from a reference or control group of apparently healthy
individuals for the
purpose of evaluation in vitro. Similarly, the expressions "reference
concentration", "reference
value", and "reference level", as used herein, refer to a value established in
a reference or control
group of apparently healthy individuals. Determination of the reference
concentration of
Arresten or Arresten expression can be made based on an amount or
concentration which best
distinguishes patient and healthy populations. By way of example, the value
for Arresten as
determined in a control group or a control population establishes a "cut-off
value" or a "reference
range". A value above such cut-off or threshold, or outside the reference
range at its higher end,
is considered to be "elevated above normal" or "diagnostic of ovarian cancer".
The reference
level can be a single number, equally applicable to every subject, or the
reference level can vary,
according to specific subpopulations of subjects. For example, post-menopausal
subjects can
have a different reference level for ovarian cancer than pre-menopausal
subjects. In addition, a
subject with more advanced ovarian cancer (e.g., stages II-IV) can have a
different reference
value than one who has early stage ovarian cancer (e.g., stage I).
[0049] As used herein, an agent "reactive" with Arresten is one that has
affinity for, binds
to, or is directed against Arresten. Such an "agent" can be a protein,
polypeptide, peptide,
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nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab')2 fragment,
molecule,
compound, antibiotic, drug, or any combination thereof. A Fab fragment is a
univalent antigen-
binding fragment of an antibody, which is produced by papain digestion. A
F(ab')2 fragment is a
divalent antigen-binding fragment of an antibody, which is produced by pepsin
digestion.
Preferably, the agent of the present invention is labeled with a detectable
marker or label.
[0050] The term "antibody", as used herein, refers to a specific protein
molecule directed
against an antigenic site and broadly includes all different types of antibody
structures, such as
monoclonal antibodies, polyclonal antibodies, multispecific antibodies
(including bispecific
antibodies), chimeric antibodies, humanized antibodies, fragments having
antigen-binding
activity, etc. For purposes of the present invention, the antibody can
specifically bind to the
biomarkers of the present invention, or the constituent proteins of the
biomarkers, and can
include polyclonal antibodies, monoclonal antibodies, and recombinant
antibodies. The
production of antibodies ¨ using, for example, Arresten as an antigen ¨ can be
performed with
techniques well known to a person of ordinary skill in the art.
[0051] The term "label" refers to a detectable compound or composition
and "labelling"
refers to the conjugation, fusion, or attachment of a detectable compound or
composition to
another. In some embodiments, the label is conjugated or fused directly or
indirectly to an agent
or reagent, such as an antibody, and assists with the detection of the agent
to which it is
conjugated or fused. The label itself can also be detectable (such as
radioisotope labels or
fluorescent labels and the like). By way of example, the label can be an
enzymatic label which
catalyzes chemical alteration of a substrate compound or composition and
results in a detectable
product.
[0052] The "sensitivity" of a biomarker, test, or assay, as described
herein, means the
probability that the biomarker, test, or assay will yield a positive result in
an individual afflicted
with cancer, particularly ovarian cancer. An increased sensitivity means fewer
false negative test
results. The "specificity" of a biomarker, test, or assay, as described
herein, means the
probability that the biomarker, test, or assay will yield a negative result in
an individual not
afflicted with cancer, particularly ovarian cancer. An increased specificity
means fewer false
positive test results.
[0053] The term "receiver operating characteristic curve" or "ROC curve"
means a
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graphical plot that illustrates the diagnostic ability of a binary classifier
system as its
discrimination threshold or cut-off is varied. In common ROC curves, the true
positive rate (TP
= sensitivity) is plotted as a function against the false positive rate (FP =
1 - specificity) for
different cut-off points for a particular biomarker or test. Each point on the
ROC curve
represents a specific sensitivity/specificity point that corresponds to a
given threshold. The area
under an ROC curve (AUC) can be a measure of how well a given biomarker or
test can
distinguish between two diagnostic outcomes. A threshold or cut-off value for
a diagnostic test
can be determined using ROC curve analysis. For example, the optimal cut-off
value can be
determined by giving equal weight to sensitivity and specificity with no
ethical, cost, and
prevalence constraints (e.g., Youden's index); alternatively, the optimal cut-
off value can be
determined by incorporating the costs of correcting false diagnosis and the
costs of further work-
up for diagnosis (e.g., utility-based decision theory). Therefore, if high
costs are involved in
false-positive diagnosis, the cut-off point can be selected at a higher value
to maximize
specificity, and if high costs are involved in false-negative diagnosis, the
cut-off point can be
selected at a lower value to maximize sensitivity.
[0054] The term "diagnosis", as used herein, refers to the identification
or classification
of a molecular or pathological state, disease, or condition (e.g., cancer, a
particular type of
cancer, etc.) and broadly includes "screening". "Diagnosis" also refers herein
to the
classification of a particular subtype of cancer, such as by histopathological
criteria or by
molecular features (including a subtype characterized by expression of one or
a combination of
biomarkers, such as particular genes or proteins encoded by the genes).
[0055] The term "prognosis", as used herein, refers to the prediction of
the likelihood of
cancer-attributable death or progression, including recurrence, metastatic
spread, and drug
resistance, of a neoplasm, such as ovarian cancer. Prognosis may also be
referred to in terms of
"aggressiveness" or "severity": an aggressive cancer is determined to have a
high risk of
negative outcome (i.e., negative or poor prognosis) and a non-aggressive
cancer has a low risk of
negative outcome (i.e., positive or favorable prognosis). An "aggressive" or
"severe" tumour is a
cell-proliferation disorder that has the biological capability to rapidly
spread outside of its
primary location or organ. Indicators of tumour aggressiveness that are
standard in the art
include, without limitation, tumour stage, tumour grade, Gleason grade,
Gleason score, nodal
13
Date Recue/Date Received 2022-06-16
status, and survival. In this context, the term "survival" is not limited to
mean survival until
mortality (wherein said mortality may be either irrespective of cause or
related to a cell-
proliferation disorder), but may also used in combination with other terms to
define clinical
outcomes (e.g., "recurrence-free survival", in which the term "recurrence"
includes both
localized and distant recurrence; "metastasis-free survival"; "disease-free
survival", in which the
term "disease" includes cancer and diseases associated therewith). The length
of the survival
may be calculated by reference to a defined starting point (e.g., time of
diagnosis or start of
treatment) and a defined end point.
[0056] The terms "treatment", "treat", "treating", and "therapy" all
refer to clinical
intervention in an attempt to alter the natural course of an individual being
treated, and can be
performed either for prophylaxis or during the course of clinical pathology.
Effects of treatment
or therapy can include preventing occurrence or recurrence of disease,
alleviating symptoms,
diminishing any direct or indirect pathological consequences of disease,
preventing metastasis,
decreasing the rate of disease progression, ameliorating the disease state,
minimizing the clinical
impairment or symptoms resulting from the disease, diminishing any pain or
discomfort suffered
by the subject, remission or improved prognosis, and extending the survival of
a subject beyond
that which would otherwise be expected in the absence of such treatment. With
reference to
cancer, "treatment" and "therapy" also include inhibiting or preventing the
development or
spread of the cancerous cells (e.g., by limiting, suspending, terminating, or
otherwise controlling
the maturation and proliferation of cells involved in the cancer).
Ovarian Cancer and Diagnosis
[0057] According to conventional thought, low-grade serous carcinoma is a
precursor
lesion for high-grade serous carcinoma. However, recent studies support the
inventor's
conclusion that ovarian carcinoma has two subsets that are molecularly
distinct as separate
diseases characterized by differing patterns of genomic variation and
prognostic implications.
Low-grade serous carcinoma (e.g., classified as type I ovarian cancer) has
better prognosis and a
more indolent/stable genomic profile, while high-grade serous carcinoma (e.g.,
classified as type
II ovarian cancer) has worse prognosis and an aggressive/distinct genomic
profile.
[0058] Molecularly, type I ovarian cancer (OC) is often characterized by
mutations in the
mitogen-activated protein kinase (MAPK) pathway (e.g., KRA,S' or BRAT). Other
significant
14
Date Recue/Date Received 2022-06-16
variants for type I OC can include alterations of genes encoding I3-catenin
(e.g., CTNNB1),
CDKN2A, PIK3CA, and PTEN which have also been found in a number of studies of
type I OC.
TP53 mutations have been rarely seen in type I OC, except in mucinous
carcinoma.
[0059] Type II OC is characterized by a high degree of
genomic/chromosomal instability,
including nearly ubiquitous mutations in TP53 that arise mainly from the
fallopian tube
epithelium. The resulting dysfunctional p53 protein or pathway is the hallmark
for the type II
OC patient group. These tumours are clinically undetectable in stage I or II,
and progress rapidly
through stages III/IV.
[0060] Diagnosis of ovarian cancer can be performed when symptoms such as
abdominal
pain (bloating), gastrointestinal (e.g., bowel) irregularities), and pelvic
pain (pressure or
discomfort) are presented in a subject at advanced stages of the disease. 30%
of patients
experience a delay in diagnosis of 2-6 months. Prior to the present invention,
OC has been
diagnosed through a positive screening test, such as a CA125 test or
transvaginal ultrasounds
(TVS). Ovarian cancer can also be diagnosed incidentally after preventive
surgery (i.e., for
BRCA carriers).
[0061] Macroscopic residual disease after debulking surgery can be an
independent
prognostic factor for survival. Among women who had undergone primary
debulking surgery,
those with no residual disease had much better seven-year survival than women
who had any
residual disease (73.6% versus 21.0%; p <0.0001) [48]). Ten-year survival can
be much
improved when surgery results in no residual disease for stage III/IV HGSOC
(50% versus 15%
for any residual disease) [49].
[0062] In OC screening, the objective is to identify an ovarian cancer at
a time when a
cure is likely (e.g., when it is possible to achieve no residual disease after
debulking). The
likelihood of achieving no residual disease diminishes with the extent of
abdominal disease the
surgeon must remove. Therefore, among women with stage III ovarian cancer, it
is desirable to
diagnose cancer as early as possible with the goal of minimizing the intra-
abdominal tumour
burden. In one study, a status of no residual disease post-surgery was
achieved for most patients
with stage IIIa/b ovarian cancer, while only about 45% of patients with stage
IIIc/IV ovarian
cancer were reduced to no residual disease [48].
Date Recue/Date Received 2022-06-16
[0063] At present, 90% of patients are diagnosed in stage IIIc/IV; only
10% are
diagnosed in stage Illa/b. In this situation, a shift of patients from stage
IIIc/IV to stage IIIa/b
has the potential to increase the probability of cure. Currently, it is almost
impossible to detect
type II OCs at their stage I/II. Most stage I/II OCs that are diagnosed are
type I OCs.
Existing Ovarian Cancer Biomarkers
[0064] To date, none of the known ovarian cancer serological biomarkers
has been
shown to be useful as a screening test in asymptomatic women because they have
poor
specificity and sensitivity in patients with early stage ovarian cancer. The
only clinical
significance of current ovarian cancer biomarkers is to differentiate between
benign and
malignant pelvic masses.
[0065] Current clinical guidelines recommend the use of biomarker CA125
for early
triage of women with pelvic masses and for the management of patients with
epithelial
ovarian/fallopian tube cancer in monitoring response to first-line
chemotherapy and post-therapy
surveillance [10]. Concentrations of CA125 greater than 95 kU/L have
discriminatory potential
for malignancy in the pelvic mass, with a positive predictive value of 95%
[11]. Persistent
elevated levels of CA125 are indicative of a poor prognosis [12].
[0066] Nevertheless, as discussed above, the specificity and sensitivity
of CA125 are far
from ideal. Among epithelial ovarian cancer patients, only 80% or so have
concentrations of
CA125 above the reference interval of 35 kU/L. Elevations have been >90% in
stages III-IV,
and 80-90% in stage II, but only 50-60% in patients with stage I OC [13,14].
[0067] Specificity of CA125 is compromised by its overexpression in
healthy pre-
menopausal women during menses and in pregnancy, as well as in some non-
malignant
gynecologic diseases (e.g., ovarian cysts, endometriosis, adenomyosis, uterine
leiomyomas),
peritoneal, pleural, and musculoskeletal inflammatory disorders, pelvic
inflammatory diseases,
and liver, renal, and cardiac diseases. Additionally, elevated concentrations
can occur in most
types of advanced adenocarcinomas, including breast, colorectal, pancreas,
lung, endometrium,
and cervix [14,15].
[0068] The frequency of CA125 overexpression is highest, though, in
serous epithelial
ovarian cancer patients, followed by endometrioid and clear cell types. CA125
is not expressed
16
Date Recue/Date Received 2022-06-16
in pure mucinous histological type of epithelial ovarian cancer [14,15].
[0069] HE4 is also found to be overexpressed in ovarian cancer [16]. It
has been shown
to have greater specificity compared with CA125, especially in the pre-
menopausal population,
in which HE4 levels are less elevated than CA125 (8% versus 29% in benign
pelvic mass);
however, HE4 still shows varying results for sensitivity [17]. HE4 is not
elevated in benign
gynecological conditions (e.g., pregnancy, menstruation, endometriosis) [18].
[0070] HE4 demonstrates the highest sensitivity for stage I diagnosis
(45.9% sensitivity
at 95% specificity) and performs better than CA125 as an indicator of worse
prognosis in
epithelial ovarian cancer [19]. The HE4 serum levels in healthy women range
from 60 pmol/L to
150 pmol/L, with higher serum levels observed in women over 40 years of age
[20,21].
Concentrations of HE4 and CA125 are the highest in endometrioid cancer (100%
overexpression) and serous epithelial ovarian cancer (93% overexpression), and
the lowest in
patients with mucinous ovarian carcinomas [22].
[0071] HE4 has also been identified in pulmonary, endometrial, and breast
carcinomas,
and mesotheliomas, but less frequently in gastrointestinal, renal, and
transitional cell carcinomas.
The most significant source of false-positive results in serum is renal
failure [23].
[0072] In 2008, Moore et al. developed the Risk of Ovarian Malignancy
Algorithm
(ROMA) that uses a combination of CA125, HE4, and menopausal status to predict
the presence
of a malignant ovarian tumour with expected higher sensitivity and specificity
compared with
CA125 alone [19,24]. Several independent prospective studies and meta-analysis
have been
carried out in order to validate the diagnostic performance of ROMA, but they
failed to reach a
clear consensus.
[0073] Van Gorp et al. performed prospective validation on 389 patients
and found that
CA125 over-performed HE4 in post-menopausal women and neither HE4 nor ROMA
improved
the diagnosis of ovarian cancer [25]. There is some limited meta-analysis-
based evidence of
ROMA performing better in early ovarian cancer and the post-menopausal
population. Other
meta-analysis studies found ROMA to have higher sensitivity, but HE4 to be
more specific.
Overall, existing meta-analysis results do not provide strong evidence for
ROMA superiority
over CA125 alone [26-29].
17
Date Recue/Date Received 2022-06-16
[0074] In 2016, the United States Food and Drug Administration (FDA)
approved a new-
generation pre-operative serum biomarker test for ovarian cancer: the OVAl
test (Overa8).
OVA1 combines 5 individual markers: CA125-II, HE4, apolipoprotein A-1,
follicle stimulating
hormone, and transferrin. Only after an ovarian mass has been determined to
require surgery is
this test then used to assess the likelihood of malignancy; thus, the use of
the OVA1 test is very
limited. OVA1 maintains a higher diagnostic sensitivity and high negative
predictive value
because five biomarkers are applied in parallel in the same serum specimen. If
the test shows
low-risk, the tumour is very unlikely to be malignant and the surgery can be
scheduled without
consulting a specialist [30].
[0075] In conclusion, CA125 is currently the only biomarker approved for
routine use in
ovarian cancer diagnosis. HE4, reporting increased specificity as compared to
CA125, requires
further validation of clinical utility. All of these biomarkers show elevation
in the late-stage
disease population, but it may not be clinically relevant. Since very high
specificities and
sensitivities are required in screening for diseases of low prevalence,
neither CA125 nor HE4
qualifies as a screening marker.
Limits to Optimizing Cut-Off for Cancer Antigen 125
[0076] Cancer Antigen 125 (CA125) is expressed on the surface of ovarian
cancer cells
and plays a role in progression and metastasis of the disease. CA125 is also
expressed in normal
epithelia of the peritoneum, in endometrium, or in benign ovarian cysts.
Various research has
been conducted to assess the levels of CA125 in different population groups.
It was reported that
1% of 883 healthy women, 6% of 143 patients with benign pelvic disease, and
82% of 102
patients with ovarian cancer showed levels of CA125 over 35 U/ml [50]. In
addition, it was
reported that 10% of benign pelvic disease (mostly pre-menopausal), 25% of
borderline OC
tumours, 40% of stage I OC tumours, and 80% of stage II-IV OC tumours showed
levels more
than 100 U/ml of CA125 [51].
[0077] It was previously reported that, among healthy individuals, 5%
showed values of
CA125 over 35 U/ml, 1% showed values over 65 U/ml, and 0.1% showed values over
100 U/ml
(seen only in pre-menopausal women) [52]. More recently, Kotsopoulos et al.
measured the
levels of CA125 of 422 patients with ovarian cancers at different disease
stages and obtained the
18
Date Recue/Date Received 2022-06-16
following results [unpublished; included here with permission]:
Stage CA125, mean (range) Median CA125 >100, n (%)1 Missing / No
CA125, n
IA (n = 47) 424.2 (6.0-5642.0) 73.0 16 (52%) 8
IB (n = 3) 23.7 (9.0-42.0) 20.0 0 0
IC (n = 44) 1013.6 (2.0-40542.0) 54.0 15 (38%) 5
IIA (n = 17) 224.9 (8.0-1298.0) 114.0 10 (63%) 1
JIB (n = 28) 556.3 (14.0-2912.0) 184.0 16 (62%) 2
IIIA (n = 16) 553.7(15.0-2191.0) 310.0 12(80%) 1
IIIB (n = 45) 1191.2 (27.0-12992.0) 516.0 36 (84%) 2
IIIC (n = 145) 1864.2 (2.0-26056.0) 719.5 125 (87%) 1
IV (n = 57) 1897.2 (54.0-11835.0) 762.5 53 (98%) 3
Missing (n = 20) 1871.2 (4.0-14450.0) 199.0 13 (81%) 4
As the above data show, if the cut-off value for CA125 is changed from 35 to
100 kU/L, more
than 80% of ovarian cancer patients with stages III and IV will be identified;
however, patients
with stage I/II may not be identified.
[0078] As discussed above, the main limitations to the use of CA125 as a
biomarker for
ovarian cancer are its low sensitivity for stage I/II and its low specificity
for pre-menopausal
women. Currently, the recommended cut-off for CA125 is 35 U/ml
internationally. Raising the
cut-off to 100 U/ml could address both of these limitations, but at the
expense of missing
borderline and stage I/II tumours, which are biologically low-risk and have a
10-year survival of
over 80%.
Arresten in Oncogenesis
[0079] Type IV collagen proteins are major structural components of
basement
membranes. The general term "matrikines" has been employed for all collagen-
derived
fragments, out of which several are known to possess anti-angiogenic activity
[42].
[0080] Arresten is the 26-kDa biologically active molecule and product of
proteolytic
cleavage derived from non-collagenous domain of type IV collagen, alpha-1
chain (encoded by
the COL4A1 gene). The base sequence of the human COL4A1 gene and the amino
acid sequence
19
Date Recue/Date Received 2022-06-16
of the encoded protein are known. For example, the nucleotide sequence of the
human COL4A1
gene and the protein amino acid sequence are registered and published in
GenBank (GenBank
Accession No. NM 001845). Arresten inhibits endothelial cell proliferation, at
least in part, by
inducing apoptotic mechanism [43].
Arresten as a Biomarker for Ovarian Cancer
[0081] The present application identifies Arresten as an ovarian cancer
biomarker that
has significantly discriminative power, without compromising on sensitivity,
before
manifestation of symptoms. Prior to the present invention, there was no study
carried out
regarding concentration of Arresten in blood, especially in connection with
early detection or
diagnosis of ovarian cancer. As detailed herein, the inventor found that
Arresten is effective as a
biomarker in ovarian cancer and its level can be used to distinguish an
ovarian cancer patient
from a normal subject.
[0082] As is well known in the art, reference levels of tumour markers
initially arise from
the need for a differentiation diagnosis between patients and healthy
population. Similarly,
serum levels of Arresten that differentiate ovarian cancer patients from
healthy or hospital
controls, who do not have ovarian cancer, can be determined. It is now
possible to develop
diagnostic methods and tools based on the use of Arresten as a biomarker for
ovarian cancer.
[0083] The area under the receiver operating characteristic (ROC) curve,
referred to as
the AUC, can be considered an appropriate measure for describing the overall
accuracy of a
biomarker or a diagnostic test. In one embodiment, the AUC value of the
Arresten biomarker for
ovarian cancer, or a diagnostic method or test relating to same, is at least
about 0.70, at least
about 0.75, at least about 0.80, at least about 0.85, or at least about 0.90.
In another embodiment,
the sensitivity of the Arresten biomarker for ovarian cancer, or a diagnostic
method or test
relating to same, is at least about 60%, at least about 65%, at least about
70%, at least about 75%,
at least about 80%, at least about 85%, or at least about 90%. In yet another
embodiment, the
specificity of the Arresten biomarker for ovarian cancer, or a diagnostic
method or test relating to
same, is at least about 75%, at least about 80%, at least about 85%, or at
least about 90%.
[0084] A specific cut-off value for the Arresten biomarker for ovarian
cancer, or a
diagnostic method or test relating to same, can be determined for use in
connection with
Date Recue/Date Received 2022-06-16
individual subjects requiring diagnosis of ovarian cancer. In one embodiment,
a threshold or cut-
off value of Arresten that is determined to be elevated above normal (e.g., a
reference
concentration or cut-off value of Arresten) can be determined to be a value
from 80 to 100 pg/ml.
For example, the threshold value of Arresten can be one of the following
concentrations in the
subject's sample (e.g., serum or plasma sample): 80 pg/ml, 85 pg/ml, 90 pg/ml,
95 pg/ml, or 100
pg/ml. The reference concentration can also be selected from the ranges of 80-
100 pg/ml, 85-
100 pg/ml, 90-100 pg/ml, and 95-100 pg/ml. In certain embodiments, the
reference
concentration is further selected from the ranges of 80-100 pg/ml, 80-95
pg/ml, 80-90 pg/ml, and
80-85 pg/ml, or from the ranges of 80-100 pg/ml and 85-95 pg/ml. In another
embodiment, the
reference concentration can be at least two times the average concentration of
Arresten in the
samples of controls (e.g., serum or plasma samples). In yet another
embodiment, the reference
concentration can be 1.2-2 times, preferably, 1.2-1.5 times, the average
concentration of Arresten
in the samples of controls (e.g., serum or plasma samples).
[0085] In addition, the following factors can also be determined: (i)
plasma levels of
Arresten, CA125, and HE4 in ovarian cancer patients at the time of diagnosis
and in healthy
women matched for menopause status; (ii) sensitivity and specificity of
Arresten in
distinguishing ovarian cancer cases from healthy controls; and (iii) whether
Arresten has a
potential to enhance specificity and sensitivity of existing biomarkers,
including CA125 and
HE4, when used in combination with them.
[0086] As one preferred approach, Arresten can be combined with different
biomarkers,
such as CA125 and HE4, in the detection, diagnosis, monitoring, and prognosis
of ovarian
cancer. In particular, Arresten can address gaps in screening, early
detection, and diagnosis of
ovarian cancer. Since Arresten is cleared from blood by the kidney, a simple
urine test for early
detection of the disease can be provided for use by women in the comfort of
their own homes
(e.g., for use periodically or if they present with a symptom associated with
ovarian cancer). As
a consequence, Arresten as a biomarker can be used in screening methods and
tools to screen the
general population for ovarian cancer. Furthermore, interaction between p53
and Arresten can
be characterized and quantified based on their expression levels in ovarian
tumour tissues.
Plasma levels of Arresten can be correlated with disease development in
ovarian cancer patients,
thereby providing new and non-invasive screening and diagnostic tests.
21
Date Recue/Date Received 2022-06-16
Diagnostic Samples
[0087] In accordance with the methods described herein, a diagnostic
sample from the
subject can be obtained using standard procedures. The diagnostic sample can
be tissue and can
be removed by standard biopsy. By way of example, the diagnostic sample can be
any tissue
known to have a neoplasm, any tissue suspected of having a neoplasm, or any
tissue believed not
to have a neoplasm. In addition, the diagnostic sample can be a bodily fluid,
including blood,
serum, plasma, vaginal secretions, urine, tears, and saliva.
[0088] Protein (e.g., Arresten) can be isolated and purified from the
diagnostic sample of
the present invention using standard methods known in the art, including,
without limitation,
extraction from a tissue (e.g., with a detergent that solubilizes the protein)
where necessary,
followed by affinity purification on a column, chromatography (e.g., FTLC and
HPLC),
immunoprecipitation (with an antibody to Arresten), and precipitation.
Isolation and purification
of the protein can be followed by electrophoresis (e.g., on an SDS-
polyacrylamide gel). In
accordance with the methods of the present invention, ovarian cancer in a
subject can be
diagnosed by assaying a diagnostic sample of the subject for expression of
Arresten, wherein
expression of Arresten elevated above normal is diagnostic of ovarian cancer.
Detecting Arresten Expression
[0089] In the methods of the present invention, a diagnostic sample of a
subject can be
assayed for Arresten expression, and Arresten expression can be detected in a
diagnostic sample,
using assays and detection methods readily determined from the known art
(e.g., immunological
techniques, hybridization analysis, fluorescence imaging techniques, and/or
radiation detection,
etc.), as well as any assays and detection methods disclosed herein (e.g.,
immunoprecipitation,
Western blot analysis, etc.). For example, a diagnostic sample of a subject
can be assayed for
Arresten expression using an agent reactive with Arresten (e.g., antibody
specific to Arresten).
[0090] The antibodies as used herein can be labeled with a detectable
marker or label.
Labeling of an antibody can be accomplished using one of a variety of labeling
techniques,
including a chemical (e.g., biotin), an enzyme (e.g., horseradish peroxidase,
alkaline
phosphatase), a radioactive material, a luminescent material, or a
chemiluminescent material
known in the art. For example, a nonradioactive or fluorescent marker, such as
biotin,
22
Date Recue/Date Received 2022-06-16
fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine, which can
be detected using
fluorescence and other imaging techniques readily known in the art.
Alternatively, the detectable
marker or label can be a radioactive marker, including, for example, a
radioisotope. The
radioisotope can be any isotope that emits detectable radiation, such as 3H,
14C, 32p, 35s,
Or
1251. Radioactivity emitted by the radioisotope can be detected by techniques
well known in the
art. For example, gamma emission from the radioisotope can be detected using
gamma imaging
techniques, particularly scintigraphic imaging. Preferably, the agent of the
present invention is a
high-affinity antibody labeled with a detectable marker or label.
[0091] Where the agent of the present invention is an antibody reactive
with Arresten, a
diagnostic sample taken from the subject can be purified by passage through an
affinity column
which contains Arresten antibody as a ligand attached to a solid support, such
as an insoluble
organic polymer in the form of a bead, gel, or plate. The antibody attached to
the solid support
can be used in the form of a column. Examples of suitable solid supports
include, without
limitation, agarose, cellulose, dextran, polyacrylamide, polystyrene,
sepharose, or other insoluble
organic polymers. The Arresten antibody can be further attached to the solid
support through a
spacer molecule, if desired. Appropriate binding conditions (e.g.,
temperature, pH, and salt
concentration) for ensuring binding of the agent and the antibody can be
readily determined by
the skilled artisan. In one embodiment, the Arresten antibody is attached to a
sepharose column,
such as Sepharose 4B.
[0092] Where the agent is an antibody, a diagnostic sample of the subject
can be assayed
for Arresten expression using binding studies that utilize one or more
antibodies immunoreactive
with Arresten, along with standard immunological detection techniques. For
example, the
Arresten molecule eluted from the affinity column can be subjected to an ELISA
analysis,
Western blot analysis, flow cytometry, or any other immunostaining method
employing an
antigen-antibody interaction.
[0093] The detection of Arresten expression in the method of the present
invention can
be followed by an assay to measure or quantify the extent of Arresten
expression in a diagnostic
sample of a subject. Such assays are well known to one of skill in the art,
and can include,
without limitation, immunohistochemistry/immunocytochemistry, flow cytometry,
mass
spectroscopy, Western blot analysis, or an ELISA for measuring amounts of
Arresten molecule.
23
Date Recue/Date Received 2022-06-16
For example, to use an immunohistochemistry assay, histological (paraffin-
embedded) sections
of tissue can be placed on slides, and then incubated with an antibody against
Arresten. The
slides then can be incubated with a second antibody (against the primary
antibody), which is
tagged to a dye or other colorimetric system (e.g., a fluorochrome, a
radioactive agent, or an
agent having high electron-scanning capacity), to permit visualization of
Arresten present in the
sections.
[0094] It is contemplated that the diagnostic sample can be assayed for
expression of any
or all forms of Arresten protein (including precursor, endoproteolytically-
processed forms, and
other forms resulting from post-translational modification) in order to
determine whether a
subject or patient has ovarian cancer. It is also contemplated that the
diagnostic sample may be
assayed for expression of Arresten elevated above normal by detecting an
increase in p53-
Arresten interaction. Accordingly, in one embodiment of the present invention,
Arresten
expression elevated above normal is detected by detecting p53-Arresten
interaction elevated
above normal.
[0095] Expected or normal levels of Arresten expression for a particular
diagnostic
sample taken from a subject or patient can be determined by assaying non-
diseased subjects of a
similar age and of the same gender. For example, diagnostic samples can be
obtained from at
least 30 normal, healthy women within a certain age range (e.g., between the
ages of 25 and 80),
to determine the normal quantity of Arresten expression in females. Once the
necessary or
desired samples have been obtained, the normal levels of Arresten expression
in women can be
determined using a standard assay for quantification, such as flow cytometry,
Western blot
analysis, or an ELISA for measuring protein quantities.
[0096] By way of example, an ELISA can be run on each sample in
duplicate, and the
means and standard deviations of the quantity of the Arresten molecule can be
determined. If
necessary, additional subjects can be recruited before the normal quantities
of Arresten
expression are quantified.
Immunoassay Detecting Arresten
[0097] Immunoassays rely on antigen-antibody interactions, in which an
antibody
inherently binds to the specific structure of its associated molecule.
Conventional immunoassay
24
Date Recue/Date Received 2022-06-16
protocols can be adapted to detect Arresten as a biomarker to diagnose ovarian
cancer in a
subject. Suitable immunoassay methods include quantitative or qualitative
immunoassay
methods conventionally developed, including Western blotting (WB),
immunofluorescence (IF),
immunocytochemistry (ICC), immunohistochemistry (IHC), immunoprecipitation
(IP), enzyme-
linked immunosorbent assay (ELISA), and flow cytometry (FCM).
[0098] All immunoassays employ a means to produce a measurable signal in
response to
antibody binding, and most involve chemically linking a detectable label to
antibodies or
antigens. Many labels are detectable because, for example, they emit
radiation, produce a color
change in a solution, fluoresce under light, or can be induced to emit light.
Detectable signals
can be measured by various detection methods, including colorimetry,
immunofluorescence
method, bioluminescence method, and chemiluminescence method.
Production of Anti-Arresten Antibody
[0099] In certain embodiments of the present invention, the agent
reactive with Arresten
is an antibody. Such an antibody can be produced using conventional methods
known in the art,
and can be polyclonal or monoclonal.
[00100] Polyclonal antibody can be produced, for example, by immunizing
any animal
species host, such as a rabbit, mouse, rat, sheep, goat, monkey, and the like,
with purified
Arresten. Monoclonal antibody then can be produced by removing the spleen from
the
immunized animal, and fusing the spleen cells with myeloma cells to form a
hybridoma which,
when grown in culture, will produce a monoclonal antibody (see, e.g., Kohler
and Milstein [53]).
Alternatively, a phage antibody library (see, e.g., Clackson et al. [54];
Marks et al. [55]) or any
other methods known in the art may be used to produce monoclonal antibody. The
antibodies of
the present invention include functional fragments of antibody molecules. A
"functional
fragment" of an antibody is an antibody fragment having antigen-binding
function.
[00101] In one embodiment, a diagnostic tool of the present invention
uses, as a reagent, a
monoclonal antibody specific to Arresten. Such an antibody can be produced by
the following
method: (i) polyclonal antibody is produced by immunizing a rabbit with
purified human
Arresten; (ii) monoclonal antibody is produced by removing the spleen from the
immunized
rabbit, and fusing the spleen cells with myeloma cells to form a hybridoma;
and (iii) the
Date Recue/Date Received 2022-06-16
hybridoma is grown in culture and the monoclonal antibody is isolated.
Types of ELBA and Their Modes of Use
[00102] As noted above, Arresten expression in a biological sample can be
detected and
quantified by ELISA. In general, there are four types of ELISA: direct,
indirect, sandwich, and
competitive.
[00103] In a direct ELISA, Arresten is immobilized directly on a solid
support (e.g., a
microplate made of polystyrene or polyvinyl chloride) in a non-specific manner
via adsorption to
the surface. A detection antibody, which is conjugated with an enzyme (e.g.,
horseradish
peroxidase (HRP), alkaline phosphatase (AP)), then binds to the immobilized
Arresten, forming
an antigen-antibody complex. Substrate for the enzyme (e.g., 3,3'5,5'-
tetramethylbenzidine
(TMB), para-nitrophenylphosphate (pNPP)) is then added, generating a signal
that is
proportional to the amount of Arresten in the sample. As an alternative, an
amplified signal can
be generated using a biotin-labelled detection antibody and an enzyme-
conjugated protein that
has an affinity to biotin, instead of the enzyme-conjugated detection
antibody. For example,
streptavidin and avidin are capable of forming a biotin complex because of
their high affinity to
biotin. While streptavidin lacks glycosylation, avidin is highly glycosylated.
These proteins can
be modified through glycosylation or deglycosylation.
[00104] In an indirect ELISA, Arresten is immobilized directly on a solid
support in the
same manner as in a direct ELISA, but the assay includes a further
amplification step for
detection. A primary detection antibody, which is unconjugated, is added and
binds to Arresten;
then a secondary detection antibody, which is conjugated with an enzyme, is
added and binds to
the primary antibody. Substrate for the enzyme is added and generates a signal
proportional to
the amount of Arresten in the sample.
[00105] In a sandwich ELISA, two antibodies specific to two different
epitopes of
Arresten are used to sandwich the protein. First, a capture antibody is coated
on a solid support.
Next, a biological sample containing Arresten is added, isolated by the
capture antibody, and
thereby specifically immobilized on the solid surface through the capture
antibody. An enzyme-
conjugated detection antibody is added and binds to an additional epitope on
Arresten, thereby
forming a complex of Arresten with the two antibodies. Substrate for the
enzyme is added and
26
Date Recue/Date Received 2022-06-16
generates a signal that is proportional to the amount of Arresten in the
sample. Sandwich
ELISAs are highly specific because two antibodies are required to bind to
Arresten.
[00106] In the sandwich ELISA described above, the detection antibody may
be
unconjugated but can itself be detected by a secondary antibody that is
conjugated to an enzyme,
in the same manner as in a indirect ELISA. Another alternative can be to
replace the enzyme-
conjugated detection antibody with a biotin-labelled detection antibody and an
enzyme-
conjugated protein that has an affinity to biotin in the same manner as in the
alternative mode of
a direct ELISA.
[00107] In a competitive ELISA, a capture antibody is coated on a solid
support in the
same manner as in a sandwich ELISA. However, instead of using an enzyme-
conjugated
detection antibody, an enzyme-conjugated antigen is used to compete for
binding with Arresten
present in a sample. Substrate for the enzyme is added and generates a signal
that is inversely
proportional to the amount of Arresten in the sample.
Exemplary Sandwich ELBA and Protocol for Arresten
[00108] Commercially available antibodies or kits can be used in a
sandwich ELISA. By
way of example, antibodies and solutions for a sandwich ELISA can be obtained
from R&D
Systems, Inc. (e.g., Human Arresten DuoSetTm ELISA, catalog# DY9925-05, R&D
Systems,
Inc., Minneapolis, MN, USA). Reagent diluents, wash buffers, or other
solutions which could
potentially impact assay performance can be routinely optimized based on type
of sample (e.g.,
serum, plasma, etc.). An exemplary sandwich ELISA is described below.
Materials
[00109] A sandwich ELISA can include the following reagents: (a) murine
capture
antibody that is specific for human Arresten; (b) murine detection antibody
that is biotin-labelled
and specific for human Arresten; (c) a human Arresten standard for calibration
(e.g., a purified
recombinant human Arresten expressed in NSO cell line); and (d) HRP-conjugated
streptavidin.
The assay is calibrated against the human Arresten standard.
[00110] The sandwich ELISA can also include: (i) 96-well microplates; (ii)
plate sealers;
(iii) phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KC1, 8.1 mM
Na2HPO4, 1.5 mM
KH2PO4, pH 7.2-7.4, 0.2 pm filtered); (iv) wash buffer (0.05% Tween 20 in
PBS, pH 7.2-7.4);
27
Date Recue/Date Received 2022-06-16
(v) reagent diluent (5% Tween 20 in PBS, pH 7.2-7.4, 0.2 p.m filtered); (vi)
substrate solution
(1:1 mixture of H202 and TMB); (vii) stop solution (2 N H2504).
Preparation of Reagents
[00111] To prepare working dilutions, all four reagents are brought to
room temperature
before use and allowed to sit for a minimum of 15 minutes with gentle
agitation after initial
reconstitution. 2.0 ml of HRP-conjugated streptavidin is diluted to a
predetermined working
concentration using reagent diluent. The capture antibody is reconstituted
with 0.5 mL of PBS
and diluted in PBS to a predetermined working concentration. The detection
antibody is
reconstituted with 1.0 mL of reagent diluent and diluted in the same to a
predetermined working
concentration. The recombinant human Arresten standard is reconstituted with
0.5 mL of
reagent diluent for assay calibration.
Preparation of Microplates
[00112] The 96-well microplates in the sandwich ELISA are coated with 100
!AL per well
of the diluted capture antibody, sealed, and incubated overnight at room
temperature. Each well
of the microplates is then aspirated and washed with wash buffer and the
process is repeated
twice for a total of three washes. Each well is washed by filling with wash
buffer (400 pL), with
liquid being completely removed at each step. After the last wash, any
remaining wash buffer is
removed. The washed microplates are then blocked by adding 300 jiL of reagent
diluent to each
well and incubated at room temperature for a minimum of an hour. The previous
aspiration /
wash step is repeated.
Assay Procedure
[00113] 100 !IL of each sample or standard diluted in reagent diluent is
added to each well
and the plates are sealed and incubated for 2 hours at room temperature. Each
well of the
microplates is aspirated and washed with wash buffer and the process is
repeated twice for a total
of three washes. Each well is washed by filling with wash buffer (400 !IL),
with liquid being
completely removed at each step. After the last wash, any remaining wash
buffer is removed.
100 jiL of the detection antibody diluted in reagent diluent is then added to
each well and the
plates are sealed and incubated for 2 hours at room temperature. The previous
aspiration / wash
step is again repeated. 100 jiL of the working dilution of EIRP-conjugated
streptavidin is then
28
Date Recue/Date Received 2022-06-16
added to each well and the plates are covered and incubated for 20 minutes at
room temperature.
The previous aspiration / wash step is again repeated. 100 [IL of substrate
solution is added to
each well and the plates are incubated for 20 minutes at room temperature. 50
[IL of stop
solution is then added to each well. The optical density of each well is
immediately determined
using a microplate reader set or corrected to one of the following
wavelengths: 450 nm, 540 nm,
or 570 nm.
[00114] A standard curve (calibration curve) is created based on a
calibration data set for
each set of samples assayed using the standard reagent (calibrator) prepared
earlier. The
calibration data set is generated at multiple concentrations or levels to
assess amount,
concentration, or level of Arresten in the sample.
[00115] For calculation of results, the duplicate readings for each
standard and sample are
averaged and the average zero standard optical density is subtracted from the
averaged duplicate
readings. Based on the foregoing, a standard curve is generated to obtain the
concentration read
for each sample.
Tests and Kits
[00116] The inventor's determination that an elevated level of Arresten
expression can be
detected in diagnostic samples from ovarian cancer patients provides new
options for identifying
patients with ovarian cancer, with the potential for commercial application in
the form of
diagnostic tools (e.g., tests, kits, etc.) for the diagnosis of ovarian cancer
and for use in general
screening procedures. Screening procedures can assist in the early detection
and diagnosis of
ovarian cancers, and can provide a method for the follow-up of patients in
whom Arresten
expression elevated above normal has been detected.
[00117] Accordingly, the present invention further provides tests and kits
for use as assays
for ovarian cancer. In some embodiments, the test or kit includes an agent
reactive with Arresten
and reagents suitable for detecting expression of Arresten. The agent can be
any of those
described above, and may be used in any of the above-described assays or
methods for detecting
or quantifying Arresten expression. Preferably, the agent of the present
invention is labeled with
a detectable marker or label.
[00118] As noted above, ELISA is one of several immunoassay methods used
to detect
29
Date Recue/Date Received 2022-06-16
and quantify specific target molecules. Three common types of ELISA are
sandwich assays,
competitive assays, and antigen-down assays. Sandwich assays are most commonly
used
because they deliver more sensitive and robust results.
[00119] In one embodiment, the diagnostic tool of the present invention is
a sandwich
ELISA capable of detecting Arresten in biological samples (e.g., blood,
plasma, urine, etc.). By
way of example, such a sandwich ELISA can include the following steps: (i)
coating an antibody
capable of binding to an epitope of Arresten, for use as a capture antibody
attached to the surface
of a solid substrate; (ii) reacting the capture antibody with the biological
sample; (iii) reacting
the resultant product of step (ii) with a detection antibody that is capable
of binding to the
complex of Arresten and the capture antibody and is labeled with a signal
generating label; and
(iv) measuring a signal originating from the label. The signal outputs can be
measured in
accordance with various methods known in the art. This detection of the signal
enables a
qualitative or quantitative analysis of the biomarker. If biotin is used as a
label, it can be easily
detected by streptavidin. When luciferase is used, luciferin can easily detect
a signal. By
analyzing the intensity of the final signal by the above-described
immunoassay, ovarian cancer
can be diagnosed. Specifically, when the signal for the biomarker in the
biological sample of a
subject appears stronger than in the normal sample, it can be determined that
the subject has
ovarian cancer.
[00120] In another embodiment, the diagnostic tool of the present
invention is a compact
point-of-care (POC) instrument employing ELISA for measuring Arresten in
blood, plasma,
serum, urine, and the like. The ELISA-based point-of-care (POC) testing may be
suitable for use
in resource-limited settings (e.g., a clinician's office) without requiring
patients to visit a
specialized laboratory for testing. The POC testing may adopt principles such
as simplification
of the procedures and miniaturization of the testing devices. Such a POC
platform may, for
example, combine the process of sandwich ELISA and the readout into a single
microfluidic chip
or cartridge, thereby providing a fully integrated, instrument-free, low-cost,
and portable device
for OC screening. Antibodies with relatively high affinity to Arresten may be
preferred for such
POC testing methods.
[00121] Due to its easy access, a POC test for ovarian cancer can be more
readily
available for screening of women with any relevant symptoms. Especially when
symptoms are
Date Recue/Date Received 2022-06-16
vague at early stages of the disease, availability of such convenient
diagnostic tools may change
the behaviors of clinicians and their patients, and result in promotion of
routine screening of the
population for ovarian cancer.
[00122] In certain embodiments, POC technology is combined with a urine
test. Urine
testing is particularly advantageous because urine samples are easily
available and can be
collected frequently in a non-invasive way. Correlation between urine and
blood levels of
Arresten can demonstrate the usefulness of urine detection of Arresten as a
surrogate for
detection of Arresten in plasma.
[00123] By way of example, a subject's urine sample may be screened for
Arresten levels
as part of a routine health examination. The test may be formatted as a urine
test strip (e.g., as a
standalone strip, cassette, or dipstick for laboratory use). A urine test
strip for identifying
whether a subject is at risk of having or has ovarian cancer may comprise a
reagent that provides
a response to the presence of Arresten when immersed in, and removed from, a
urine sample of
the subject; such a response indicates whether the subject is at risk of
having or has ovarian
cancer.
[00124] The urine test strip may be produced by means known to a person
skilled in the
art. In one embodiment, the urine test strip is provided in a device for
testing a urine sample of a
subject. The urine test strip may be prepared with a pad or matrix of an
absorbent material. A
labeled antibody specific to Arresten may also be provided in an area on the
urine strip, so that
urine, the labeled antibody, and Arresten flow together through the absorbent
material by
capillary action. The strip can be prepared from any suitable material through
which the urine
test sample, any Arresten therein, and labeled antibody can flow by
capillarity. Suitable matrix
materials include, without limitation, nitrocellulose, polysulfones,
polycarboxylic acids, and
filter paper.
[00125] In another embodiment, a urine test device may contain an
immunoassay strip, an
absorbent matrix or pad, and a plastic housing. The immunoassay strip can be
formed by
compressing nonwoven fibers into a narrow strip and coating them at least
partially with a
reactive antibody. In use, the antibody of the device may bind to Arresten
present in a urine
sample, ultimately resulting in a color change. The absorbent pad or matrix
may extend to
contact the urine stream of a user. The absorbent matrix can absorb the liquid
and draw it into
31
Date Recue/Date Received 2022-06-16
contact with the immunoassay strip. The immunoassay strip and the absorbent
pad may be
contained within a 2-piece housing that allows the unit to be handheld and
protects the strip from
environmental contaminants. A leak-proof, clear plastic window on the side of
the housing can
prevent urine from accidentally splashing on the immunoassay strip and permit
the test and
control zone portions of the strip to be viewed.
[00126] In still another embodiment, a strip-based ELISA test, similar to
a pregnancy test,
can be used by individual women at their own homes to detect Arresten in urine
samples. This
can promote ovarian cancer screening for even asymptomatic women, obviating
the need to go to
a doctor's office or laboratory for testing.
[00127] As noted above, ovarian cancers are associated with high fatality,
but early
detection can significantly improve survival rates. Convenient urine tests for
use in detecting
Arresten as a biomarker in ovarian cancer may promote early detection in
patients, thereby
increasing survival rates.
Exemplary Methods
[00128] In accordance the present invention, Arresten as an ovarian cancer
biomarker can
be detected, alone or in combination with other markers such as CA125 and HE4,
in a biological
sample. As discussed above, the cut-off value for CA125 can be increased
(e.g., to 95-100 U/m1)
in order to increase sensitivity of the biomarker for late-stage ovarian
cancer (e.g., stage III or
IV), but at the expense of detecting patients with early stage ovarian cancer
(e.g., stage I or II).
Combining Arresten detection with CA125 detection (with an increased cut-off
value) may
compensate for detecting stage I/II patients. Therefore, in one embodiment, a
diagnostic sample
of a subject is assayed for detecting the expression of both Arresten and
CA125 to diagnose
ovarian cancer in the subject. In certain preferred embodiments, the cut-off
value for CA125 is
80-120 kU/L, 85-110 kU/L, 90-105 kU/L, or 95-100 kU/L. More preferably, the
cut-off value
for CA125 is 95 kU/L or 100 kU/L.
[00129] The present invention further provides a method for assessing the
efficacy of
therapy to treat ovarian cancer in a subject or patient who has undergone or
is undergoing
treatment for ovarian cancer. The method includes assaying a diagnostic sample
of the subject or
patient for Arresten expression, where detection of a normal level of Arresten
expression is
32
Date Recue/Date Received 2022-06-16
indicative of successful therapy to treat ovarian cancer, and detection of
Arresten expression
elevated above normal is indicative of not responding to treatment. The
diagnostic sample can
be a tissue or a bodily fluid, as described above, and can be assayed for
expression of Arresten.
In addition, the diagnostic sample can be assayed for expression of Arresten
using all of the
various assays and methods of detection and quantification described above.
[00130] This method of the present invention provides a means for
monitoring the
effectiveness of therapy to treat ovarian cancer by permitting the periodic
assessment of levels of
Arresten expression in a diagnostic sample taken from a subject or patient. In
accordance with
this method, a diagnostic sample of a subject or patient can be assayed, and
levels of Arresten
expression can be assessed, at any time following the initiation of therapy to
treat ovarian cancer.
For example, levels of Arresten expression can be assessed while the subject
or patient is still
undergoing treatment for ovarian cancer. Where levels of Arresten expression
detected in an
assayed diagnostic sample of the subject or patient continue to remain
elevated above normal, a
physician can choose to continue with the subject's or patient's treatment for
the cancer or change
it to a treatment to which tumour cells better respond. Where levels of
Arresten expression in an
assayed diagnostic sample of the subject or patient decrease through
successive assessments, it
can be an indication that the treatment for ovarian cancer is working, and
that treatment doses
could be decreased or even ceased. Where levels of Arresten expression in an
assayed diagnostic
sample of the subject or patient do not rapidly decrease through successive
assessments, it can be
an indication that the treatment for ovarian cancer is not working, and that
treatment doses could
be increased or the choice of treatment could be changed.
[00131] It is within the confines of the present invention to assess
levels of Arresten
expression following completion of a subject's or patient's treatment for
ovarian cancer, in order
to determine whether the cancer has recurred in the subject or patient.
Accordingly, an
assessment of levels of Arresten expression in an assayed diagnostic sample
can provide a
convenient way to conduct follow-ups of patients who were previously diagnosed
with ovarian
cancer. Furthermore, it is within the confines of the present invention to use
assessed levels of
Arresten expression in an assayed diagnostic sample as a clinical or
pathologic staging tool, as a
means of determining the extent of ovarian cancer in the subject or patient,
and as a means of
ascertaining appropriate treatment options.
33
Date Recue/Date Received 2022-06-16
[00132] A correlation exists, in general, between tumour burden and the
survival of a
patient who has cancer, and, more specifically, between pelvic mass and an
ovarian cancer
patient. Therefore, it is also contemplated herein that assaying a diagnostic
sample of a subject
for Arresten expression can be a useful means of providing information
concerning the prognosis
of a subject or patient who has ovarian cancer. Accordingly, the present
invention further
provides a method for assessing the prognosis of a subject who has ovarian
cancer, comprising
assaying a diagnostic sample of the subject for Arresten expression, where the
subject's
prognosis improves with a decrease in Arresten expression in the diagnostic
sample of the
subject, and the subject's prognosis worsens with an increase in Arresten
expression in the
diagnostic sample of the subject.
[00133] According to the method of the present invention, a diagnostic
sample of a subject
or patient can be assayed, and levels of Arresten expression can be assessed,
at any time during
or following the diagnosis of ovarian cancer in the subject or patient. For
example, levels of
Arresten expression in an assayed diagnostic sample can be assessed before the
subject or patient
undergoes treatment for ovarian cancer, in order to determine the subject's or
patient's initial
prognosis. Additionally, levels of Arresten expression in an assayed
diagnostic sample can be
assessed while the subject or patient is undergoing treatment for ovarian
cancer, in order to
determine whether the subject's or patient's prognosis has become more or less
favorable through
the course of treatment.
[00134] By way of example, where levels of Arresten expression detected in
an assayed
diagnostic sample of the subject or patient are, or continue to remain,
significantly high, a
physician can conclude that the subject's or patient's prognosis is
unfavorable. Where Arresten
expression in an assayed diagnostic sample of the subject or patient decreases
through successive
assessments, it can be an indication that the subject's or patient's prognosis
is improving. Where
levels of Arresten expression in an assayed diagnostic sample of the subject
or patient do not
decrease significantly through successive assessments, it can be an indication
that the subject's or
patient's prognosis is not improving. Finally, where Arresten expression is
low, or is normal, in
a diagnostic sample of the subject or patient, a physician can conclude that
the subject's or
patient's prognosis is favorable.
[00135] The present invention also provides methods of treating a subject
who has been
34
Date Recue/Date Received 2022-06-16
diagnosed with ovarian cancer using Arresten as a biomarker. The standard
treatment for
ovarian cancer currently consists of debulking surgery followed by six rounds
of chemotherapy.
In one embodiment, a diagnostic sample of a patient is analyzed for Arresten
expression, and a
therapy is provided to the subject when the Arresten expression is elevated
above normal.
Exemplary therapies include, without limitation, surgical debulking,
chemotherapy, or radiation
therapy, and any combination of the foregoing, including the current standard
treatment of
debulking surgery followed by chemotherapy. Typical chemotherapy drugs combine
a platinum-
based drug (e.g., carboplatin or cisplatin) with a taxane (e.g., paclitaxel or
docetaxel).
[00136] The present invention is described in the following Examples,
which are set forth
to aid in an understanding of the invention, and should not be construed to
limit in any way the
scope of the invention as defined in the claims which follow thereafter.
EXAMPLES
EXAMPLE 1 ¨ STUDY SUBJECTS I
[00137] Plasma samples derived from ovarian cancer patients (n = 22) and
healthy
controls (n = 20) were tested for Arresten levels by an enzyme-linked
immunosorbent assay
(ELISA).
[00138] The ovarian cancer cases were obtained from the Poland Ovarian
Cancer Study
(POC). The POC study consisted of women with familial ovarian cancer who were
referred to 1
of 16 clinical cancer genetics centers throughout Poland between 1999 and
2001. These centers
were established in 1998 as a national network with support from the Polish
Ministry of Health
for the purpose of coordinating cancer genetics services. Eligible women
included those with
invasive ovarian cancer and at least one first- or second-degree relative with
ovarian cancer
diagnosed at any age or with early-onset breast cancer (diagnosed at age 50 or
below). Of those,
22 ovarian cancer patients with high-grade serous type were randomly selected
for this study
before they started treatment.
[00139] The control women were those who participated in mammography
screening for
their routine screening tests at 8 different centers all over Poland between
2009 and 2011 and
who provided blood samples for DNA analysis. Women with breast or ovarian
cancer were
Date Recue/Date Received 2022-06-16
excluded from this group; 20 healthy women were randomly selected for the
study.
EXAMPLE 2¨ LABORATORY ASSAY I
[00140] Plasma Arresten was quantified using a laboratory-made ELISA. All
plasma
samples were run in duplicate. The Arresten concentration was calculated as
the average of
duplicate samples (each adjusted for background signal and normalized to blank
wells). The
average intra-assay coefficient of variation (CV) was approximately 1.7%. This
was calculated
using the mean CV of duplicate samples. A non-parametric Mann-Whitney U test
was used for
comparing the mean Arresten plasma levels in plasma of the cases and the
controls.
EXAMPLE 3¨ DETECTION OF ARRESTEN IN URINE SAMPLES
[00141] The same ELISA method of Example 2 that was used for detecting
Arresten
levels in plasma was also used to measure Arresten in urine samples of four
healthy individuals.
It was shown that Arresten is detectable in urine and its urine level
generally correlates with the
matched plasma level.
[00142] A urine-based biomarker test facilitates screening and diagnosis
processes due to
non-invasiveness of the collection method and the feasibility of developing
ELISA paper strips
to be used by women periodically for testing for ovarian cancer in the comfort
of their own
homes.
EXAMPLE 4¨ VALIDATION STUDY I
[00143] 400 plasma samples are collected from ovarian cancer patients with
different
histology, and 400 control samples collected from healthy subjects. Ovarian
cancer plasma
samples are obtained from Princess Margaret Hospital's Ovarian Cancer Biobank
in Toronto,
Canada. The control plasma samples are obtained from the biobank at Women's
College
Hospital in Toronto. From each patient, 300 jil of plasma is used for
measuring three
biomarkers: Arresten, HE4, and CA125. Three sections (10 microns each) from
ovarian-tumour
formalin-fixed paraffin-embedded (FFPE) blocks are obtained from 20 randomly-
selected
patients for measuring p53 and Arresten expression in tumour tissues.
[00144] Arresten levels are determined by enzyme-linked immunosorbent
assay (ELISA).
36
Date Recue/Date Received 2022-06-16
The specificity and sensitivity of Arresten as a biomarker are determined,
alone and in
combination with HE4 and CA125 ¨ other known biomarkers which are also
measured by
ELISA.
[00145] The expression levels of Arresten and p53 in ovarian tumour
tissues are measured
by immunohistochemistry (IHC) assay.
[00146] In order to determine diagnostic performance and optimal cut-off
values for
Arresten plasma levels, the sensitivities, specificities, and area under a
receiver operating
characteristic curve (AUC) are calculated. Similar calculations are performed
for CA125 and
HE4 and for combined biomarkers. The significance of two-group comparisons are
calculated
using a Mann-Whitney non-parametric test. Biomarker composite scores are
calculated by
logistic regression from standardized biomarker values.
[00147] The power of this study is more than 90% to detect a 10%
difference in mean of
measured biomarkers between patient cases and controls, based on 400 samples
in each group.
EXAMPLE 5¨ STUDY SUBJECTS II
[00148] For Validation Study II (Example 7), the inventor examined plasma
samples from
421 unselected ovarian cancer patients with different histology and 407
hospital control patients.
The hospital controls were non-cancer patients with urinary incontinence (252)
or myoma (153).
Ovarian cancer and control plasma samples were obtained from hospitals in
Szczecin, Poland
affiliated with Pomeranian Medical School. These samples were collected from
ovarian cancer
patients at the time of diagnosis between 2019 and 2021. The controls were
women referred to
the hospitals for non-malignant issues. Controls matched cases for age and
menopausal status.
The individuals in the study consented to participate in a biobank for
studying ovarian cancer.
Patients' characteristics and clinical data were collected at the time of
diagnosis.
EXAMPLE 6¨ LABORATORY ASSAY II
[00149] Arresten levels in the study subjects of Example 5 were determined
by sandwich
ELISA (enzyme-linked immunosorbent assay). Human Arresten DuoSetTm ELISA
(Catalog#
DY9925-05) from R&D Systems, Inc. (Minneapolis, MN, USA) was routinely
optimized for
plasma samples in the inventor's lab at Women's College Hospital and used in
the study. All
37
Date Recue/Date Received 2022-06-16
measurements were made in duplicate and samples with a coefficient of
variability (CV) of more
than 15% were excluded from the study.
EXAMPLE 7¨ VALIDATION STUDY II
[00150] The plasma levels of Arresten were measured and compared among the
study
subjects (421 ovarian cancer cases and 407 hospital control women) of Example
5. The
significance of two-group (cases and controls) comparisons were calculated
using either the
Mann-Whitney non-parametric test where the distributions were not normal or a
Student's t-test
where the distributions were normal. Adjustment for confounding variables and
further analysis
were conducted by logistic regression. Biomarker performance was determined by
calculating
the AUC ¨ the area under the receiver operating characteristic (ROC) curve.
The optimal cut-
off, sensitivity, and specificity of the biomarker were also determined by
analyzing the ROC
curve.
[00151] As discussed in greater detail below, the data showed that plasma
levels of
Arresten were significantly higher in ovarian cancer patients than in controls
and could have use
as a biomarker for diagnosis and tumour staging of ovarian cancer. This
study's power to detect
a 10% difference in the mean of measured biomarkers between cases and controls
based on over
400 samples in each group was more than 90%.
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION FOR EXAMPLES 1 AND 2
[00152] The mean plasma level of Arresten was significantly higher in
ovarian cancer
patients than in healthy controls (1020 ng/ml versus 446 ng/ml; p = 0.0001).
This represents a
two-fold higher plasma level among ovarian cancer patients, which is opposite
to what was
expected. The higher concentration of Arresten in the plasma of ovarian cancer
patients makes
this protein even more favorable as a screening or diagnostic biomarker,
despite the contrary
hypothesis that linked Arresten with defective p53 function in ovarian
cancers. One explanation
for the contrary observation could be that Arresten is expected to be
triggered by high expression
levels of p53 even in its mutated, non-functional form. Preferably, it could
be determined
whether ovarian cancer patients tested in Examples 1 and 2 also carry high
expression levels of
38
Date Recue/Date Received 2022-06-16
the mutant p53 protein.
RESULTS AND DISCUSSION FOR EXAMPLES 5-7
[00153] Table 1 summarizes the details of plasma levels of Arresten
measured in the 421
ovarian cancer patients and the 407 hospital controls who were the study
subjects of Example 5:
Table 1. Statistics of Arresten levels in control and case samples.
Group Mean (pg/ml) Median (pg/ml) p-Value (comparison with
control)
Control 66.27 57.76 Reference
All Cases 247.95 115.97 <0.001
Stage I 89.31 61.07 0.5
Stage II 114.64 90.29 <0.001
Stage III 258.07 170.46 <0.001
Stage IV 526.99 350.69 <0.001
As the results show, the ovarian cancer patients had a mean Arresten level of
247.95 pg/ml,
which was significantly higher than the mean Arresten level of the controls
(66.27 pg/ml; p <
0.001) (see Figure 1; Table 1).
[00154] Comparing the ovarian cancer patients with the controls at each
tumour stage, the
Arresten levels of patients with stage I tumours showed no significant
difference with the
hospital controls (mean: 89.31 pg/ml versus 66.27 pg/ml; p = 0.5). However,
plasma
concentrations of Arresten in patients with stage II, III, or IV tumours were
significantly higher
than the hospital controls (mean for stage II = 114.64 pg/ml, mean for stage
III = 258.07 pg/ml,
mean for stage IV = 526.99 pg/ml versus hospital controls = 66.27 pg/ml; p
<0.001) (see Figure
1; Table 1).
[00155] With reference to Figure 1, Arresten levels were significantly
higher in the
ovarian cancer cases than in the controls (p <0.001). When controlling for
stage, Arresten levels
of the ovarian cancer cases at stage I were not significantly different from
the controls (p = 0.5).
However, the difference between cases and controls in stages II-IV was
significant (p <0.001).
39
Date Recue/Date Received 2022-06-16
[00156] With reference to Figure 2, analyzing the receiver operating
characteristic (ROC)
curve revealed an AUC of 0.745, with an optimal sensitivity of 64.6% and
specificity of 76.63%
at a cut-off value of 78.9 pg/ml (at specificity = 90%, sensitivity = 32.8%).
[00157] With reference to Figure 3, the ROC curve for the stage II
patients resulted in an
AUC of 0.713, with an optimal sensitivity of 60.7% and specificity of 78.8% at
a cut-off value of
81.88 pg/ml (at specificity = 90%, sensitivity = 7.1%) (see Figure 3A). The
ROC curve for the
stage III patients resulted in an AUC of 0.865, with an optimal sensitivity of
80.4% and
specificity of 80.2% at a cut-off value of 83.96 pg/ml (at specificity = 90%,
sensitivity = 41.8%)
(see Figure 3B). The ROC curve for the stage IV patients resulted in an AUC of
0.900, with an
optimal cut-off value of 121.75 pg/ml, sensitivity of 80.5%, and specificity
of 94.6% (at
specificity = 90%, sensitivity = 69.5%) (see Figure 3C).
[00158] As the foregoing results reveal, Arresten plasma levels were
higher in ovarian
cancer cases than in hospital non-cancer controls (p < 0.001), and levels
increase with tumour
stage. Arresten levels at stage I of ovarian cancer were not significantly
different from controls.
This was not unexpected, because Arresten is released due to extracellular
matrix breakdown
during metastasis ¨ which, in the case of ovarian cancer, starts from stage
II. However, after
stage I, Arresten levels increased as cancer progressed through the various
stages, and its
performance as a diagnostic biomarker was improved in advanced stages, as
shown by the
increasing AUC of the ROC analysis of stages II to IV (see Figures 1 and 3).
[00159] Overall, Arresten showed a decent performance as an ovarian cancer
biomarker
with a sensitivity of 64.6% and specificity of 76.63% (see Figure 2). Arresten
performance was
improved at advanced stages; however, its levels were significantly higher in
ovarian cancer
cases than in controls as early as stage II. These results support use of
Arresten as a potential
diagnostic biomarker for ovarian cancer, and use of plasma levels of Arresten
as an indicator of
tumour stage.
ENUMERATED EMBODIMENTS
[00160] The invention is further exemplified by the following enumerated
non-limiting
embodiments that would be understood by those skilled in the art to be merely
exemplary of the
methods, uses, kits, and other implementations consistent with the description
herein.
Date Recue/Date Received 2022-06-16
Numbered Embodiments ¨ Example A
1. A method for determining whether a subject has ovarian cancer,
comprising assaying a
diagnostic sample of the subject for Arresten expression, wherein detection of
Arresten
expression elevated above normal is diagnostic of ovarian cancer in the
subject.
2. The method of embodiment 1, wherein Arresten expression elevated above
normal is
detected by detecting p53-Arresten interaction elevated above normal.
3. The method of embodiment 1, further comprising the step of obtaining the
diagnostic
sample from the subject.
4. The method of embodiment 1, wherein the ovarian cancer is an epithelial
carcinoma.
5. The method of embodiment 4, wherein the epithelial carcinoma is a
serous, mucinous,
endometrioid, clear cell, transitional cell, squamous cell, or mixed
epithelial neoplasm.
6. The method of embodiment 1, wherein the ovarian cancer is a p53-
associated ovarian
cancer.
7. The method of embodiment 1, wherein the ovarian cancer is primary
ovarian cancer.
8. The method of embodiment 1, wherein the ovarian cancer is high-grade
serous ovarian
cancer.
9. The method of embodiment 1, wherein the diagnostic sample is a serum,
plasma, or urine
sample.
10. The method of embodiment 1, wherein the diagnostic sample is assayed
using an agent
reactive with Arresten.
11. The method of embodiment 10, wherein the agent is an antibody or an
antigen-binding
fragment thereof.
12. The method of embodiment 10 or 11, wherein the agent is labeled with a
detectable
marker.
13. The method of embodiment 1, wherein the diagnostic sample is assayed
using an ELISA,
a chemiluminescence assay, or an immunohistochemistry assay.
14. The method of embodiment 13, wherein the ELISA is an ELISA paper strip.
41
Date Recue/Date Received 2022-06-16
15. The method of embodiment 1, wherein the detected Arresten expression is
at least two
times higher than normal.
16. The method of embodiment 1, wherein the Arresten expression is elevated
above normal
in the diagnostic sample when the Arresten expression is above a concentration
selected from
400 to 1000 ng/ml.
17. The method of embodiment 16, wherein the diagnostic sample is plasma.
18. The method of embodiment 1, wherein the subject is at least one of the
following:
(a) pre-menopausal;
(b) asymptomatic of ovarian cancer;
(c) not carrying a mutation of BRCA/ or BRCA2 gene;
(d) suffering from a non-malignant gynecologic disease, a peritoneal,
pleural, or
musculoskeletal inflammatory disorder, a pelvic inflammatory disease, a liver,
renal, or cardiac
disease, or an advanced adenocarcinoma.
19. The method of embodiment 1, wherein the ovarian cancer is a type II
ovarian cancer.
20. The method of embodiment 19, wherein the type II ovarian cancer is at
stage I, II, Ma, or
TIM.
21. The method of embodiment 1, wherein the ovarian cancer is undetectable
by a CA125
test or a transvaginal ultrasound test.
22. The method of embodiment 1, further comprising assaying the diagnostic
sample of the
subject for at least one additional biomarker selected from CA125 and HE4.
23. The method of embodiment 22, wherein the at least one additional
biomarker is CA125
with a cut-off value of 95-100 U/ml.
24. A method of screening a general population for ovarian cancer,
comprising: testing a
plurality of asymptomatic subjects in accordance with the method of embodiment
1.
25. A method for treating ovarian cancer in a subject, comprising:
(a) analyzing a diagnostic sample of the subject for Arresten expression;
and
(b) providing a therapy to the subject when the Arresten expression is
elevated above
normal,
42
Date Recue/Date Received 2022-06-16
wherein the therapy comprises at least one of surgical debulking,
chemotherapy,
radiation, and hormone therapy.
26. The method of embodiment 25, wherein the ovarian cancer is high-grade
serous ovarian
cancer.
27. The method of embodiment 26, wherein the high-grade serous ovarian
cancer is at stage
I, II, Ma, or IIIb.
28. A method for determining the molecular subtype of ovarian cancer in a
patient who has
been diagnosed with ovarian cancer, comprising:
(a) assaying a biological sample of the patient for Arresten expression;
and
(b) either:
(i) determining that the patient has type I ovarian cancer when the
Arresten
expression in the biological sample is normal, or
(ii) determining that the patient has type II ovarian cancer when the
Arresten
expression in the biological sample is elevated above normal.
29. The method of embodiment 28, wherein:
detection of Arresten expression elevated above normal is indicative of type
II ovarian
cancer in which p53 in ovarian cancer cells is mutated, and
detection of normal Arresten expression is indicative of type I ovarian cancer
in which
p53 in ovarian cancer cells is not mutated.
30. A method for assessing the severity of ovarian cancer in a patient who
has been
diagnosed with ovarian cancer, comprising:
(a) assaying a biological sample of the patient for Arresten expression
prior to
treatment; and
(b) either:
(i) determining that the patient has a favorable prognosis when Arresten
expression in the biological sample is normal, or
(ii) determining that the patient has a poor prognosis when Arresten
expression in the biological sample is elevated above normal.
31. The method of embodiment 30, wherein:
43
Date Recue/Date Received 2022-06-16
detection of Arresten expression elevated above normal in the biological
sample is
indicative of ovarian cancer in which p53 in the ovarian cancer cells is
mutated, and
detection of normal Arresten expression in the biological sample is indicative
of ovarian
cancer in which p53 in the ovarian cancer cells is not mutated.
32. A method for assessing the efficacy of therapy to treat ovarian cancer
in a subject who
has undergone or is undergoing treatment for ovarian cancer, comprising:
(a) assaying a first diagnostic sample of the subject for Arresten
expression after
therapy has commenced;
(b) obtaining a first level of Arresten expression in the first diagnostic
sample; and
(c) comparing the first level of Arresten expression with a second level of
Arresten
expression in a second diagnostic sample of the same subject, wherein the
second diagnostic
sample was assayed and the second level was obtained prior to the therapy,
wherein a significant decrease of the first level of Arresten expression,
relative to the
second level of Arresten expression, indicates that the subject is responding
to the therapy to
treat ovarian cancer, and a minor or no decrease of the first level of
Arresten expression, relative
to the second level of Arresten expression, indicates that the subject is not
responding to the
therapy.
33. The method of embodiment 32, wherein the first level of Arresten
expression is below a
concentration selected from 400-1000 ng/ml, and the second level of Arresten
expression is
above the selected concentration.
34. A kit for use in detecting ovarian cancer, comprising:
(a) an agent reactive with Arresten; and
(b) at least one reagent suitable for detecting expression of Arresten.
35. The kit of embodiment 34, which is adapted for ELISA-based point-of-
care testing.
36. The kit of embodiment 35, further comprising a second agent reactive
with CA125 and at
least one second reagent suitable for detecting expression of CA125.
37. The kit of embodiment 36, wherein:
the Arresten cut-off value is 400-1000 ng/ml, preferably 446-1020 ng/ml, and
the CA125 cut-off value is 95-100 U/ml.
44
Date Recue/Date Received 2022-06-16
38. A device for detecting ovarian cancer in a subject, comprising:
(a) a housing;
(b) a matrix of absorbent material for contacting a urine sample of the
subject; and
(c) a strip of immunoassay at least partially coated with a labeled anti -
Arresten
antibody for detecting Arresten in the subject's urine sample,
wherein the absorbent matrix and the immunoassay strip are arranged within the
housing
in a manner such that the absorbent matrix, upon contact with the urine
sample, draws the liquid
into the immunoassay strip by capillary action.
39. The device of embodiment 38, wherein the absorbent material comprises
nitrocellulose,
polysulfones, polycarboxylic acids, or filter paper.
40. Use of an antibody specific to Arresten for the diagnosis, treatment,
or prognosis of
ovarian cancer in a subject.
41. Methods comprising any features, combinations of features, and/or sub-
combinations of
features described herein.
42. Kits comprising any features, combinations of features, and/or sub-
combinations of
features described herein.
43. Devices comprising any features, combinations of features, and/or sub-
combinations of
features described herein.
44. Uses of any features, combinations of features, and/or sub-combinations
of features
described herein.
Numbered Embodiments ¨ Example B
1. A method for diagnosing ovarian cancer in a human female subject,
comprising:
isolating Arresten that is present in a diagnostic sample of the subject by
immobilizing
the Arresten on a solid surface;
forming a complex of the immobilized Arresten with a primary antibody specific
for
Arresten, said complex coupled with an enzyme to form an enzyme complex;
incubating the enzyme complex with a substrate for the enzyme;
measuring a detectable signal produced by the enzyme acting on the substrate;
and
Date Recue/Date Received 2022-06-16
calculating a level of Arresten in the diagnostic sample based on the
measurement of the
detectable signal,
wherein the level of Arresten elevated above a predetermined cut-off value is
diagnostic
of ovarian cancer in the subject.
2. The method of embodiment 1, wherein the enzyme in the enzyme complex is
conjugated
to at least one of the following:
(a) the primary antibody;
(b) a secondary antibody that binds to the primary antibody; and
(c) a protein that binds to a biotin labelling: (i) the primary antibody or
(ii) the
secondary antibody.
3. The method of embodiment 2, wherein the protein is streptavidin or
avidin, and wherein
the streptavidin or avidin is native or modified by glycosylation or
deglycosylation.
4. The method of any one of embodiments 1-3, wherein the immobilized
Arresten is directly
immobilized to the solid surface or indirectly immobilized to the solid
surface through a capture
antibody that is coated on the solid surface and specifically binds to
Arresten.
5. The method of any one of embodiments 1-4, further comprising comparing
the detectable
signal to a calibration data set generated using a calibrator at multiple
concentrations or levels to
assess amount, concentration, or level of Arresten in the diagnostic sample,
wherein the
calibrator is recombinant human Arresten.
6. The method of any one of embodiments 1-5, wherein the detectable signal
is measured by
colorimetry or by a method selected from the group consisting of
immunofluorescence,
bioluminescence, and chemiluminescence.
7. The method of any one of embodiments 1-6, wherein:
the predetermined cut-off value is 80-100 pg/ml; or
the predetermined cut-off value is set so that the method has: (a) a
sensitivity of at least
about 60%; (b) a specificity of at least about 75%; or (c) a sensitivity of at
least about 60% and a
specificity of at least about 75%.
8. The method of any one of embodiments 1-7, wherein the predetermined cut-
off value is
set so that the method has a specificity or sensitivity of about 90% or
greater.
46
Date Recue/Date Received 2022-06-16
9. The method of embodiment 7, wherein the predetermined cut-off value is
determined
based on a receiver operating characteristic (ROC) curve with an area under
the curve (AUC) of
at least about 0.70.
10. The method of embodiment 9, wherein the AUC is at least about 0.85.
11. The method of embodiment 9, wherein the predetermined cut-off value is
based on at
least one of the following: (a) a type of ovarian cancer and (b) a stage of
ovarian cancer.
12. The method of embodiment 11, wherein the predetermined cut-off value is
specific to
stage II ovarian cancer and set so that the method has a sensitivity of least
about 60%.
13. The method of embodiment 12, wherein the subject is asymptomatic of
ovarian cancer.
14. The method of any one of embodiments 1-13, wherein the diagnosing is to
screen the
subject for ovarian cancer, who is asymptomatic of ovarian cancer.
15. A method for treating ovarian cancer in a subject, comprising:
administering to the subject a treatment comprising at least one of surgical
debulking,
chemotherapy, and radiation therapy, wherein the subject was diagnosed with
ovarian cancer by
a process comprising:
(a) obtaining a serum or plasma sample from the subject;
(b) analyzing the sample for Arresten expression; and
(c) detecting Arresten expression elevated above normal, thereby diagnosing
the
subject with ovarian cancer.
16. The method of embodiment 15, wherein the process has a sensitivity of
at least about
60% and a specificity of at least about 75%.
17. The method of embodiment 16, wherein the process is based on a receiver
operating
characteristic (ROC) curve with an area under the curve (AUC) of at least
about 0.70.
18. A diagnostic kit for assessing risk of ovarian cancer in a female
subject by measuring a
level of Arresten expression in a biological sample obtained from the subject,
the kit comprising:
(a) a capture antibody that is capable of specifically binding to human
Arresten,
thereby isolating human Arresten from the biological sample;
(b) a solid matrix to which the capture antibody will bind;
47
Date Recue/Date Received 2022-06-16
(c) a detection antibody that is capable of specifically binding to human
Arresten and
has a label for generating a detectable signal;
(d) a recombinant human Arresten standard for calibration; and
(e) at least one reagent suitable for generating the detectable signal in
cooperation
with the label, thereby detecting a level of human Arresten in the biological
sample.
19. The diagnostic kit of embodiment 18, further comprising:
(a) a second capture antibody that is capable of specifically binding to
human
CA125, thereby isolating human CA125 from the biological sample;
(b) a second detection antibody that is capable of specifically binding to
human
CA125, and has a second label for generating a second detectable signal;
(c) a recombinant human CA125 standard for calibration; and
(d) at least one second reagent suitable for generating the second
detectable signal in
cooperation with the second label, thereby detecting expression of human
CA125.
20. The diagnostic kit of embodiment 19, wherein:
a predetermined cut-off value of the human Arresten is 80-100 pg/ml, and
a predetermined cut-off value for the human CA125 is 35-100 U/ml.
21. The diagnostic kit of any one of embodiments 18-20, which is adapted
for point-of-care
testing.
22. The method of any one of embodiments 1-14, further comprising the step
of obtaining the
diagnostic sample from the subject.
23. The method of any one of embodiments 1-17, wherein the ovarian cancer
is an epithelial
carcinoma.
24. The method of embodiment 23, wherein the epithelial carcinoma is a
serous, mucinous,
endometrioid, clear cell, transitional cell, squamous cell, or mixed
epithelial neoplasm.
25. The method of any one of embodiments 1-17, wherein the ovarian cancer
is primary
ovarian cancer.
26. The method of any one of embodiments 1-17, wherein the ovarian cancer
is high-grade
serous ovarian cancer.
48
Date Recue/Date Received 2022-06-16
27. The method of any one of embodiments 1-14, wherein the diagnostic
sample is a serum,
plasma, or urine sample.
28. The method of any one of embodiments 1-14, comprising use of a paper-
based ELISA.
29. The method of any one of embodiments 1-14, wherein the predetermined
cut-off value is
1.2-2 times greater than normal level.
30. The method of any one of embodiments 1-17, wherein the subject is at
least one of the
following:
(a) pre-menopausal;
(b) asymptomatic of ovarian cancer;
(c) not carrying a mutation of BRCA/ or BRCA2 gene;
(d) suffering from a non-malignant gynecologic disease, a peritoneal,
pleural, or
musculoskeletal inflammatory disorder, a pelvic inflammatory disease, a liver,
renal, or cardiac
disease, or an advanced adenocarcinoma.
31. The method of any one of embodiments 1-17, wherein the ovarian cancer
is a type II
ovarian cancer.
32. The method of embodiment 31, wherein the type II ovarian cancer is at
stage II, Ma, or
IIIb.
33. The method of any one of embodiments 1-17, wherein the ovarian cancer
is undetectable
by a CA125 test or a transvaginal ultrasound test.
34. The method of any one of embodiments 1-14, further comprising assaying
the diagnostic
sample of the subject for at least one additional biomarker selected from
CA125 and HE4.
35. The method of embodiment 34, wherein the at least one additional
biomarker is CA125
with a predetermined cut-off value of 35-100 U/ml.
36. An in vitro method for using Arresten as a biomarker for screening a
human female
subject for ovarian cancer, comprising:
exposing a diagnostic sample of the subject to a surface that is coated with a
first anti-
Arresten antibody;
adding a second anti-Arresten antibody, wherein the first and second
antibodies bind to
49
Date Recue/Date Received 2022-06-16
different epitopes of Arresten;
measuring an amount of the second antibody bound to Arresten isolated from the
diagnostic sample of the subject; and
determining a level of Arresten in the diagnostic sample based on the amount
of the
second antibody,
wherein the level of Arresten elevated above a predetermined cut-off value is
indicative
of ovarian cancer in the subject.
37. A method of preparing an Arresten-antibody complex for diagnosing and
treating ovarian
cancer in a human female subject, comprising:
obtaining a serum or plasma sample from the subject;
providing a capture antibody and a detection antibody that specifically bind
to Arresten,
wherein the capture and detection antibodies bind to different epitopes of
Arresten;
coating a support surface with the capture antibody and incubating at room
temperature;
adding the sample to the support surface coated with the capture antibody,
thereby
contacting the capture antibody with Arresten present in the sample, and
incubating at room
temperature;
adding the detection antibody to Arresten bound to the capture antibody and
incubating at
room temperature; and
producing a complex of Arresten with the capture and detection antibodies on
the support
surface for detection of an elevated level of Arresten expression in the
subject.
38. An Arresten-antibody complex for diagnosing and treating ovarian cancer
in a human
female subject, prepared in accordance with the method defined in embodiment
37.
39. A method for administering an ovarian-cancer therapy to a subject,
comprising:
(a) directing a clinical laboratory to
(i) process a serum or plasma sample of the subject with a reagent containing
an
antibody that binds to Arresten;
(ii) measure a level of Arresten expression in the sample; and
(iii) communicate the measured level of Arresten; and
(b) administering an ovarian-cancer therapy comprising at least one of
surgical
debulking, chemotherapy, and radiation therapy when the measured level is
elevated as
Date Recue/Date Received 2022-06-16
compared with a reference sample.
40. A method for diagnosing and treating ovarian cancer in a subject,
comprising:
obtaining a serum or plasma sample from the subject;
detecting Arresten expression in the sample elevated above normal, thereby
diagnosing
the subject with ovarian cancer, wherein the elevated Arresten expression is
detected using an
immunoassay including at least one reagent that is an anti-Arresten antibody
capable of forming
an antigen-antibody complex with Arresten present in the sample, and
administering to the subject a treatment comprising at least one of surgical
debulking,
chemotherapy, and radiation therapy.
41. A method of screening a general population for ovarian cancer,
comprising: testing a
plurality of asymptomatic subjects in accordance with the method of embodiment
1.
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All publications mentioned herein are hereby incorporated by reference in
their entireties. While
the foregoing invention has been described in some detail for purposes of
clarity and
understanding, it will be appreciated by one skilled in the art, from a
reading of the disclosure,
that various changes in form and detail can be made without departing from the
true scope of the
invention in the appended claims.
Date Recue/Date Received 2022-06-16
