BIOMARKERS FOR THE DIAGNOSIS OF PROSTATE CANCER IN A NON-HYPERTENSIVE POPULATION

BIOMARQUEURS POUR LE DIAGNOSTIC DU CANCER DE LA PROSTATE CHEZ DES PATIENTS NON HYPERTENDUS

English Abstract

The present invention includes the use of biomolecules for differential
diagnosis of
prostate cancer and/or non-malignant disease of the prostate. The present
invention also
provides methods for detecting biomolecules within a biological sample. The
invention
further includes kits for differential diagnosis of prostate cancer and/or non-
malignant
disease of the prostate in a biological sample.

Claims

We claim:
1. A method of diagnosing prostate cancer in a subject, comprising:
(a) detecting a quantity, presence, or absence of PSP94 in a first biological
sample from
the subject;
(b) optionally detecting total PSA in the first biological sample or a second
biological
sample from the subject; and
(c) comparing the quantity, presence or absence of PSP94 and optionally total
PSA as
detected in steps (a) and (b) with a standard score.
2. The method of diagnosing prostate cancer in a subject of claim 1, wherein
said standard
score is obtained from one or more subjects known to have prostate cancer,
wherein a
similarity in quantity, presence, or absence of PSP94 and optionally total PSA
between the
quantity, presence or absence as detected in steps (a) and (b) with said
standard score
results in a diagnosis of prostate cancer in the subject.
3. The method of diagnosing prostate cancer in a subject of claim 1, wherein
said standard
score is obtained from one or more known subjects having a Gleason score of
less than or
equal to 6, wherein a deviation in quantity, presence, or absence of PSP94 and
optionally
total PSA between the quantity, presence or absence as detected in steps (b)
and (c) with
said standard score results in a diagnosis of aggressive prostate cancer in
the subject.
4. The method of diagnosing prostate cancer in a subject of claim 1, wherein
said standard
score is obtained from one or more subjects known to have a Gleason score of
greater than
or equal to 7, wherein a similarity in quantity, presence, or absence of PSP94
and
optionally total PSA between the quantity, presence or absence as detected in
steps (b) and
(c) with said standard score results in a diagnosis of aggressive prostate
cancer in the
subject.
5. The method of diagnosing prostate cancer in a subject of claim 1, wherein
said standard
score is obtained from one or more subjects known to be selected from the
group
consisting of (i) healthy subjects, (ii) subjects having a precancerous
prostatic lesion, (iii)
subjects with non-malignat disease of the prostate, (iv) subjects with
localized cancer of
40

the prostate, (v) subjects having an acute or chronic inflammation of
prostatic tissue (v)
subjects with metastasised cancer of the prostate, wherein a similarity or
difference
between the quantity, presence or absence of PSP94 and optionally the total
PSA in the
first or the first and second biological samples and the standard score is
used to determine
whether the subject is healthy or has a precancerous prostatic lesion, a non-
malignant
disease of the prostate, a localized cancer of the prostate, an acute or
chronic inflammation
of prostatic tissue, or a metastasised cancer of the prostate.
6. The method of diagnosing prostate cancer in a subject of claim 1, wherein
said standard
score is obtained from the subject in the past, wherein a deviation in
quantity, presence, or
absence of PSP94 and optionally total PSA between the quantity, presence or
absence as
detected in steps (a) and (b) with said standard score results in a indicator
of the
progression of the prostate cancer in the subject.
7. The method of any one of claims 1-6 further comprising the step of
diagnosing whether
the subject has hypertension.
8. The method of any one of claims 1-6 wherein the subject does not have
hypertension.
9. The method of any one of claims 1-8 wherein the detection of the quantity,
presence or
absence of PSP94 and optionally total PSA comprises the steps of:
(a) contacting the biological sample with a biologically active surface; and
(b) allowing the PSP94 and optionally PSA within the biological sample to bind
to the
biologically active surface.
10. The method of any one of claims 1-8 wherein the detection of the quantity,
presence
or absence of PSP94 and optionally total PSA comprises the steps of:
(a) contacting the biological sample with one or more binding molecule
specific for PSP94
and PSA; and
(b) detecting the quantity, presence or absence of PSP94 and optionally PSA.
11. The method of any one of claims 1-10 wherein the subject is diagnosed as
having
aggressive prostate cancer when the quantity of PSA is determined to be
between 0.0 - 10
ng/ml.
41

12. The method of any one of claims 1-11 wherein the quantity, presences or
absence of
PSP94 and total PSA are detected by utilizing an antibody specific to PSP94 or
total PSA.
13. The method of any one of claims 1-12 wherein the quantity, presence, or
absence of
PSP94 and total PSA are detected by utilizing an ELISA assay.
14. The method of any one of claims 1-13 wherein the quantity, presence, or
absence of
PSP94 and total PSA are detected through use of a multiplex immunoassay.
15. The method of any one of claims 1-14 wherein the biological sample is
selected from
the group consisting of whole blood, blood serum, blood plasma, urine, semen,
seminal
fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's
fluid), excreta, tears,
saliva, sweat, biopsy, ascites, cerebrospinal fluid, lymph, and a biopsy
sample.
16. The method of claim 15 wherein the biological sample is urine.
17. The method of any one of claims 1-16 wherein the sample is collected by
spot
collection.
18. The method of any one of clams 1-17 wherein the sample is collected by 24
hour
collection.
19. A kit for diagnosing prostate disease in a subject comprising: a
biologically active
surface comprising an adsorbent, binding solutions, and instructions to use
the kit; wherein
the instructions outline a method for diagnosis of a prostate cancer in a
subject according
to the invention or a method for the differential diagnosis of healthy, non-
malignant
disease of the prostate, precancerous prostatic lesion, localized cancer of
the prostate,
metastasised cancer of the prostate, and acute or chronic inflammation of
prostatic tissue
in a subject according to the method of any one of claims 1-19.
20. The kit of claim 19 comprising a biologically active surface comprising an
adsorbent
comprised of silicon dioxide molecules.
42

21. The kit of claim 20 comprising a biologically active surface comprising an
adsorbent
comprising antibodies specific to PSP94 and optionally PSA.
22. A method of computing a standard score, the method comprising:
receiving with programmable electronics a first input identifying a first
characteristic of a subject, the first input including a PSA value;
receiving with the programmable electronics a second input identifying a
second
characteristic of the subject, the second input including a PSP94 value; and
computing with the programmable electronics a standard score using the first
and
second characteristics of the subject and storing the standard score in a
memory device.
23. The method of claim 22, further comprising determining if the subject has
hypertension, and computing the standard score only if the subject does not
have
hypertension.
24. The method of claim 22, further comprising:
receiving a blood pressure input; and
determining whether the subject has hypertension using the blood pressure
input;
and;
computing the standard score only if determined that the subject does not have
hypertension.
25. The method of claim 24, further comprising prompting the user for the
first and
second inputs only determined that the subject does not have hypertension.
26. The method of claim 22, wherein the programmable electronics include at
least one
processor and the least one memory device.
27. The method of claim 26, wherein the programmable electronics include at
least
two processors.
28. The method of claim 27, wherein the at least two processors are in data
communication across a data communication network.
43

29. The method of claim 22, wherein receiving the first and second inputs
comprises
receiving the first and second inputs with an input device of a computing
device.
30. The method of claim 22, further comprising displaying the standard score
on a
display device of the programmable electronics, wherein the programmable
electronics
include a computing device.
31. The method of claim 22, further comprising saving the standard score in a
medical
record of a subject in a medical records database.
32. The method of claim 22, further comprising sending the standard score to a
computing device across a network.
33. The method of claim 22, wherein computing a standard score comprises
calculating the results of a mathematical formula.
34. The method of claim 33, wherein the mathematical formula is based on
fitting a set
of data to a logit function logistic curve using logistic regression.
35. The method of claim 33, wherein the set of data includes data for a
plurality of
subjects, the data including at least a PSA value and a PSP94 value for each
subject.
36. The method of claim 22, wherein computing a standard score comprises
computing
a result of:
y = A log(PSA) + B log(PSP94) + C
where y is the standard score; PSA is the PSA value; PSP94 is the PSP value;
and A, B,
and C are constants.
37. The method of claim 36, wherein A is 2.2724, B is -1.4732, and C is
0.3839.
38. The method of claim 22, further comprising determining if the subject has
hypertension, and computing the standard score only if the subject does not
have
hypertension.
44

39. The method of claim 22, wherein the standard scare is adapted to he used
for
subject diagnosis.
40. The method of claim 22, wherein the standard score is adapted to be used
for
prostate cancer diagnosis.
41. A computer-readable storage medium comprising instructions that, when
executed
by a computer, cause the computer to:
receive a first input identifying a first characteristic of a non-hypertensive
subject,
the first input including a PSA value;
receive a second input identifying a second characteristic of the subject, the
second
input including a PSP94 value; and
compute with a computing device a standard score using the first and second
characteristics of the subject and storing the standard score in a memory
device.
42. The computer-readable storage medium of claim 22, wherein the first input
is
received with the computing device with an input device.
43. The computer-readable storage medium of claim 22, wherein the first input
is
received from a second computing device.
44. A method of diagnosing a subject, the method comprising:
determining if the subject has hypertension;
if the subject does not have hypertension, receiving a biological sample from
the
subject;
determining a first characteristic of the subject from the biological sample,
the first
characteristic including a PSA value;
determining a second characteristic of the subject from the biological sample,
the
second characteristic including a PSP94 value;
computing a standard score based at least in part on the PSA value and the
PSP94
value; and
diagnosing prostate cancer in the subject using the standard score.

45. The method of claim 44, wherein computing the standard score comprises
computing a result of:
y= A log(PSA) +B log(PSP94) + C
where y is the standard score; PSA is the PSA value; PSP94 is the PSP94 value;
and A, B,
and C are constants.
46. The method of claim 45, wherein A is 2-2724,B is -1.4732, and C is 0.3839.
47. A system comprising:
at least one processor; and
memory, the memory storing instructions that, when executed by the processor,
cause the processor to:
receive a first input identifying a first characteristic of a non-hypertensive
subject, the first input including a PSA value;
receive a second input identifying a second characteristic of the subject, the
second input including a PSP94 value; and
compute with a computing device a standard score using the first and
second characteristics of the subject and storing the standard score in a
memory
device.
46

Description

CA 02747184 2011-07-22
131OMARKIRS FOR THE DIAGNOSIS OF PROSTATE CANCER IN A NON-
IIYPERTENSIVE POPULATION
FIELD OF THE INVENTION
The present invention relates to diagnosing prostate cancer. More
particularly, the present
invention includes a method for differential diagnosis of prostate cancer from
a non-malignant
disease of the prostate and/or from a healthy prostate.
BACKGROUND
Prostate cancer is one of the most common cancers to afflict men in western
countries. In
North America the incidence rate for prostate cancer in males is an estimated
166.7 per 100,000
per year, which accounted for an estimated 33% of all newly reported cancers
in men in 2005
(American Cancer Society, 2005)_ The Canadian Cancer Society indicates that
one in 7 men will
develop prostate cancer, mostly after.age 70 (Canadian Cancer Society, 2005).
In 2005, American
Cancer Society and Canadian Cancer Society estimated the mortality rate for
this disease to be
20% (American Cancer Society, 2005; Canadian Cancer Society, 2005)_
The current standard screening method for prostate cancer is the Prostate
Specific Antigen
(PSA) test, which can take the form of total PSA measurements, free-total PSA
ratios, and PSA
velocities (change in PSA levels over time) (Egawa. et al., 1997; Djavan et
al., 1999). Typically,
an individual has been characterized as having an elevated risk for prostate
cancer with a PSA
level above 4.0 ng/mL (Gann et al., 1995). This can be refined to account for
a number of factors,
such as PSA levels increasing naturally with age (Oesterling et al., 1994).
Clinicians must rely on
complementary diagnostic tools because PSA screening is an imperfect means of
diagnosis, is not
indicative of pathological stage (Bedusehi and Oesterling, 1997; Erdem et al.,
2002-2003), and has
poor specificity. The result is healthy patients being subjected to
unnecessary testing and an
increased financial and emotional toll of prostate cancer diagnosis. The
primary diagnostic tools
used in addition to PSA testing are the digital-rectal exam (DRE) and prostate
biopsy- DREs are
performed routinely in conjunction with PSA tests and biopsies to improve the
accuracy of.
diagnosis (Scattoni et al., 2003). Prostate biopsies are the means of ultimate
confirmation of
diagnosis, but have significant complication rates (Rodriguez and Terris,
1998). The U.S.
Preventative Services Task Force does not recommend the PSA test for routine
screening. Despite
the known shortcomings of PSA testing and significant amounts of research,
there has been little
improvement in the state of the prostate disease diagnostics. Thus, there is
an unmet need for
more accurate prostate disease diagnostics, particularly prostate cancer.
Prostate, Secretory Protein (PSP94), also known as beta-microseminoprotein or
inhibin-
like peptide. is a basic 94 amino acid protein with a molecular weight of
10,704 (Seidah et al.).
PSP94 is generated from a 114 amino acid precursor encoded by a nucleotide
sequence located on

CA 02747184 2011-07-22
chromosome 10 (Dube et al, 1987). Purified PSP94 isolated from seminal fluid
migrates between
13 - 16 kDa on a polyacryliinide gel (Dube et al, 1987), and the difference in
molecular weight is
not due to glycosylation but due to the basic nature of the protein (Seidab at
at, 1984). PSP94 is
found in high concentration in prostate epithelial cells (Brar et al, 1988)
and has been examined in
serum and urine for its potential to be a cancer biomarkcr. Results from
Kaighn et al. (1987)
demonstrated that PSP94 was not detectable in a PC-3 cell line from human
prostatic carcinomas.
furthermore, PSP94 in urine was decreased in men with late stage tumors using
24 hr collection
(Teni at al, 1988; Teni Cl a!, 1989; Trembley et al, 1987).
Abnormal protein levels in serum are indicative of prostate cancer, wherein
the irregular or
erratic control of PSP94 secretion from the prostate is correlated with
neoplasia (Wu at al-, 1999).
Several groups explored PSP94 for the potential of becoming a relevant
biomarker for prostate
cancer (Dubs et al., 1987; Tremblay et al., 1987; Abrahamsson at al., 1988;
Teni at al., 1988;
Abrahamsson ct aL, 1989; Teni et al., 1989; von der Kammer et al., 1990; Huang
at al., 1993;
1lyakutake et al., 1993; von der Kammer et al., 1993, Maeda at at., 1994;
Tsunisaki at al., 1998;
Sakai at al., 1999). However, these groups were were never successful at
producing a reliable
diagnostic test for prostate cancer utilizing PSP94.
SUMMARY OF THE INVENTION
Recently, we identified a peak at 10750 M/Z by mass spectrometry that
decreased in the
urine of patients with prostate cancer- The peak corresponded to PSP94. We
them developed an
immunoassay to measure PSP94 in urine to develop a commercial assay to help
minimize the
number of prostate biopsies for men with PSA values between 0.0 and 10.0
ng/ml, with a negative
DRE.
An aspect of the present invention relates to methods for differential
diagnosis of prostate
cancer or non-malignant disease of the prostate by detecting PSP94 and PSA
within a test sample
of a given subject, comparing results with samples from healthy subjects,
subjects having
precancerous prostatic lesion, subjects with non-malignant disease of the
prostate, subjects with
localized cancer of the prostate, subjects with metastasised cancer of the
prostate, and/or subjects
with an acute or a chronic inflammation of prostatic tissue, wherein
comparison allows for
differential diagnosis of a subject as healthy, having a precancerous
prostatic lesion, having non-
malignant disease of the prostate, having localized prostate cancer, having a
metastasised prostate
cancer or having an acute or chronic inflammation of prostatic tissue. In an
embodiment, the
subject does not have hypertension.
One aspect of the invention includes a method for diagnosing prostate cancer
in a subject
comprising detecting a quantity, presence, or absence of PSP94 and total PSA
in a biological
sample; and classifying said subject as having or not having prostate cancer,
based on said
quantity, presence or absence of PSP94 and total PSA. In one embodiment, the
step of classifying
2

CA 02747184 2011-07-22
said subject comprises comparing the quantity, presence, or absence of PSP94
and total PSA with
a reference value indicative of a prostate cancer. In an embodiment, the
subject does not have
hypertension.
A further aspect of the invention includes a method for differential diagnosis
of prostate
cancer and non-malignant disease of the prostate in a subject, comprising
detecting a quantity,
presence or absence of PSP94 and total PSA in a biological sample and
classifying said subject as
having prostate cancer, non-malignant disease of the prostate, or as healthy,
based on the quantity,
presence or absence of PSP94 and total PSA in said biological sample. in one
embodiment, the
step of classifying said subject comprises comparing a quantity, presence, or
absence of PSP94 and
total PSA with a reference value indicative of prostate cancer and a reference
value indicative of a
non-malignant disease of the prostate. in an embodiment, the subject does not
have hypertension.
A further aspect of the invention includes a method for differential diagnosis
ofhealtby,
non-malignant disease of the prostate, precancerous prostatic lesion,
localized cancer of the
prostate, metastasised cancer of the prostate, and acute or chronic
inflammation of prostatic tissue
in a subject, comprising detecting a quantity, presence or absence of PSP94
and total PSA in a
biological sample and classifying said subject as having non-malignant disease
of the prostate,
precancerous prostate lesion, localized cancer of the prostate, metastasised
cancer of the prostate,
and/or acute or chronic inflammation of prostatic tissue, or as healthy, based
on the quantity,
presence or absence of PSP94 and total PSA in said biological sample. In one
embodiment, a step
of classifying said subject comprises comparing a quantity, presence or
absence of PSP94 and total
PSA with a reference value indicative of healthy, non-malignant disease of the
prostate,
precancerous prostate lesion, localized cancer of the prostate, metastasised
cancer of the prostate,
acute inflammation of prostatic tissue or chronic inflammation of prostatic
tissue. In an
embodiment, the subject does not have hypertension. In a further aspect
diagnosis includes
differential diagnosis.
An aspect of the present invention relates to methods for evaluating a
prognosis of prostate
cancer in a subject. The methods comprise detecting a quantity of PSP94 and
total PSA in a test
sample; and classifying the progression of cancer. The present method permits
differentiation of
prostate cancer subjects with a good prognosis (high probability of recovery,
becoming disease
free) from subjects with a bad prognosis (low probability of recovery, cancer
reoccurrence,
metastasis)- in an embodiment, the subject does not have hypertension.
In a further embodiment of the methods of the invention, a quantity, presence,
or absence
of PSP94 and total PSA are detected or quantified in a biological sample
obtained from the subject
utilizing an antibody to said biomarker.
In a further embodiment of the methods of the invention, a quantity, presence,
or absence
of PSP94 and total PSA are detected or quantified in a biological sample
obtained from the subject
through the use of an ELISA assay.
3

CA 02747184 2011-07-22
In a further embodiment of the methods of the invention, a quantity, presence,
or absence
of PSP94 and total PSA are detected or quantified in a biological sample
obtained from the subject
through the use of a BioPlex"9 Immunoassay (Bio-Rad Laboratories, Hercules,
CA).
In a further embodiment of the methods of the invention, a quantity, presence,
or absence
of PSP94 and total PSA are detected or quantified through a use of a biochip.
In a further embodiment of the invention, a quantity, presence, or absence of
PSP94 and
total PSA are detected or quantified in an automated system.
In a further embodiment of the invention, a subject is a mammal. The subject
may be a
human.
In a further embodiment of the invention, a test or biological sample used
according to the
invention may be blood, blood serum, blood plasma, urine, semen, seminal
fluid, seminal plasma,
prostatic fluid, prc-ejaculatory fluid (Cowper's fluid), excreta, tears,
saliva, sweat, bile, biopsy,
ascites, cerebrospinal fluid, lymph, or tissue extract origin. In a further
embodiment of the
methods of the invention, the test and/or biological samples are urine, semen,
seminal fluid,
seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid)
samples, and are isolated
from subjects of mammalian origin, preferably of human orig~in_ In a still
further embodiment of
the invention, the test and/or biological samples are blood, blood serum,
plasma and/or urine.
In a further embodiment of the invention the biological sample can be urine.
In a further embodiment of the present invention the urine can be collected
from the
patient by spot collection or 24 hour collection.
In a further embodiment of the invention, a biologically active surface
comprises an
adsorbent comprising silicon dioxide molecules.
A further aspect of the invention includes a kit for diagnosing prostate
disease within a
subject comprising: a biologically active surface comprising an adsorbent,
binding solutions, and
instructions to use the kit, wherein the instructions outline a method for
diagnosis of a prostate
cancer in a subject according to the invention or a method for the
differential diagnosis of healthy,
non-malignant disease of the prostate, precancerous prostatic lesion,
localized cancer of the
prostate, metastasised cancer of the prostate, and acute or chronic
inflammation of prostatic tissue
in a subject according to the invention- In an embodiment, the subject does
not have hypertension.
in an embodiment of the invention, a kit comprises a biologically active
surface
comprising an adsorbent comprised of silicon dioxide molecules.
In an embodiment of the invention, a kit comprises a biologically active
surface
comprising an adsorbent comprising antibodies specific to PSP94 and PSA.
A further aspect of the invention includes a method for in vitro diagnosis of
a prostate
cancer in a subject comprising detecting PSP94 and total PSA in a biological
sample by- (a)
contacting a biological sample from a subject with one or more binding
molecule specific for
PSP94 and PSA and (b) detecting a quantity, presence or absence of PSP94 and
PSA, and
4

CA 02747184 2011-07-22
determining PIT PSA, in the sample, wherein a. quantity, presence or absence
of PSP94 and total
PSA allows for diagnosis of the subject as healthy or having prostate cancer.
In an embodiment,
the subject does not have hypertension-
A further aspect of the invention includes a method for in vitro differential
diagnosis of
prostate cancer and non-malignant disease of the prostate in a subject,
comprising detecting PSP94
and total PSA in a biological sample: (a) contacting a biological sample with
a binding molecule
specific for PSP94 and PSA; and (b) detecting a quantity, presence or absence
of PSP94 and PSA,
in the sample, wherein the quantity, presence or absence of PSP94 and total
PSA allows for the
differential diagnosis of the subject as having prostate cancer, and/or having
a non-malignant
disease of the prostate, or as being healthy. In an embodiment, the subject
does not have
hypertension.
In an embodiment according to the invention for in vitro diagnosis of prostate
cancer in a
subject, for in vitro differential diagnosis of prostate cancer and non-
malignant disease of the
prostate in a subject, or for in vitro differential diagnosis of healthy,
prostate cancer, non-
malignant disease of the prostate, precancerous prostatic lesion, localized
cancer of the prostate,
metastasised cancer of the prostate, and acute or chronic inflammation of
prostatic tissue in a
subject, detection is performed by an immunosorbent assay. In an embodiment,
the subject does
not have hypertension.
A further aspect of the invention comprises a kit for diagnosis of a prostate
disease within
a subject comprising a binding solution, one or more binding molecule(s), a
detection substrate,
and instructions, wherein the instructions outline a method according to the
invention for in vitro
diagnosis of prostate cancer in a subject, for in vitro differential diagnosis
of prostate cancer and
non-malignant disease of the prostate in a subject, or for in vitro
differential diagnosis of healthy,
prostate cancer, non-malignant disease of the prostate, precancerous prostatic
lesion, localized
cancer of the prostate, metastasised cancer of the prostate, and acute or
chronic inflammation of
prostatic tissue in a subject. In an embodiment, the subject does not have
hypertension.
A further aspect of the invention comprises a use of PSP94 and total PSA for
differential
diagnosis of non-malignant disease of the prostate, precancerous prostatic
lesion, localized cancer
of the prostate, metastasised cancer of the prostate or acute or chronic
inflammation of prostatic
tissue.
A further aspect of the invention comprises a use of the detection or
quantification of
PSP94 and total PSA in a biological sample from a subject for determination of
whether the
subject has prostate cancer. In an embodiment, the subject does not have
hypertension.
A further aspect of the invention comprises a use of the detection or
quantification of
PSP94 and total PSA in a biological sample from a subject for determination of
whether the
subject has non-malignant disease of the prostate. in an embodiment, the
subject does not have
hypertension.
5

CA 02747184 2011-07-22
A further aspect of the invention comprises a use of the detection or
quantification of
PSP94 and total PSA in a biological sample from a subject for determination of
whether the
subject has benign prostate disease, precancerous prostalie lesions, localized
cancer of the prostate,
metastasised cancer of the prostate, or acute or chronic inflarnmation of the
prostate- In an
embodiment, the subject does not have hypertension.
A further aspect of the invention comprises a use of PSP94 and total PSA to
detect
prostate cancer.
A further aspect of the invention includes a method of identifying a molecular
entity that
inhibits or promotes an activity of PSP94 and total PSA according to the
invention comprising: (a)
selecting a control animal having PSP94 and total PSA and a test animal having
PSP94 and total
PSA; (b) treating the test animal using the molecular entity or a library of
molecular entities, under
conditions to allow specific binding and/or interaction, and (c) determining a
relative quantity of
PSP94 and total PSA, as between the control animal and the test animal. In an
embodiment of the
invention, animals are mammals. Mammals may be rats, mice, or primates.
A further aspect of the invention includes a method of identifying a molecular
entity that
inhibits or promotes an activity of PSP94 and total PSA comprising: (a)
selecting a host cell
expressing PSP94 and PSA; (b) cloning the host cell and separating the clones
into a test group
and a control group; (c) treating the test group using the molecular entity or
a library of molecular
entities under conditions to allow specific binding and/or interaction, and
(d) determining a relative
quantity of PSP94 and total PSA, as between the test group and the control
group.
A further aspect of the invention includes a method for identifying a
molecular entity that
inhibits or promotes an activity of PSP94 and total PSA comprising: (a)
selecting a test group
having a host cell expressing PSP94 and total PSA and a control group; (b)
treating the test group
using the molecular entity or a library of molecular entities; and (c)
determining a relative quantity
of PSP94 and F/T PSA, as between the test group and the control group.
In an embodiment of the invention, a host cell is a neoplastic or cancer cell.
In an embodiment of any of the methods according to the invention for
identifying a
molecular entity that inhibits or promotes an activity of PSP94 and total PSA,
a library of
molecular entities can be nucleotides, oligonucleotides, polynucleotides,
amino acids, peptides,
polypeptides, proteins, antibodies, immunoglobulins, small organic molecules,
pharmaceutical
agents, agonists, antagonists, derivatives, and/or combinations thereof.
A further aspect of the invention includes a composition for treating a
prostate disease
comprising a molecular entity, which modulates PSP94 and total PSA and a
pharmaceutically
acceptable carrier.
An embodiment of the invention includes a composition for treating a prostate
disease
selected from the group consisting of prostate cancer and non-malignant
disease of the prostate.
6

CA 02747184 2011-07-22
A further embodiment includes a composition for treating a prostate disease
selected from
the group consisting of non-malignant disease of the prostate, precancerous
prostatic lesion,
localized cancer of the prostate, metastasised cancer of the prostate, and
acute or chronic
inflammation of prostatic tissue.
A further embodiment of the invention includes a composition comprising a
molecular
entity that can be polynucleotides, amino acids, polypeptides, small organic
molecules,
pharmaceutical agents, or combinations thereof The polypeptides can be
antibodies, agonists,
antagonists, derivatives, or combinations thereof.
A further aspect of the invention includes a composition for treating prostate
disease
comprising a molecular entity identified by any one of the methods of
invention for identifying a
molecular entity, which inhibits or promotes the activity of PSP94 and/or PSA,
and a
pharmaceutically acceptable carrier.
In an embodiment of the invention, a composition comprises a molecular entity
that is
comprised of polynucleotides, amino acids, peptides, polypeptides, proteins,
small organic
molecules, pharmaceutical agents, agonists, antagonists, derivatives or
combinations thereof
A further aspect of the invention includes a use of any composition according
to the
invention for treating a prostate disease. Prostate disease may be prostate
cancer and non-
malignant disease of the prostate. The prostate disease may be is selected
from the group
consisting of non-malignant disease of the prostate, precancerous prostatic
lesion, localized cancer
of the prostate, metastasised cancer of the prostate, and acute or chronic
inflammation of prostatic
tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure I is a flow chart illustrating an example method of diagnosing a
subject.
Figure 2 is a flow chart illustrating an example method of computing a
standard score
based on at least first and second characteristics of a subject. '
Figure 3 is a schematic block diagram illustrating an architecture of an
example
computing device for implementing various aspects according to the present
disclosure.
Figure 4 is a ROC Curve for the diagnosis of BPI=I compared the prostate
cancer by
PSP94 concentrations in pre-DRE spot urine collection (ng/mL), and PSP94 in 24
hr urine
collection (ng/mL).
Figure 5 is a box and whisker Plot of PSP94 24 hr categorized by Gleason
Score. PSP94
in 24 hr collection is in ng/mL and Gleason Score 0 = non cancer samples. The
upper and lower
box is 25% and 75% of data respectively, the line within the box is the median
and the whiskers
are 5% and 95% limits of the data. Points that are shaded are outliers.
Figure 6 is a ROC curve for Logistic Regression model for PSP94 spot
collection, PSA,
PSP94 spot collection for noncancer compared to HGS samples in men without
hypertension.
7

CA 02747184 2011-07-22
Figure 7 is a ROC curve for PSP94 spot collection, 24 hr collection and PSA
for men
without hypertension and PSA values below 10 nglmL. AUC is 0.923.
DETAILED DESCRIPTION OF THE INVENTION
The term "biomolecule" refers to a molecule that is produced by a cell or
tissue in an
organism. Such molecules include, but are not limited to, molecules comprising
polynucleotides,
amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, fatty
acids, lipids, steroids,
and combinations thereof (e.g., glycoproteins, ribonucleoproteins,
lipoproteins)- The terms
"nucleotide", "oiigonueleotide" or polynucleotide" refer to DNA or RNA or
synthetic origin which
may be single-stranded or double-stranded and may represent the sense or the
antisense strand.
Included as part of the definition of "oligonucleotide" or "polynueleotide"
are peptide
polynueleotide sequences (i.e. peptide nucleic acids; PNAs), or any DNA-like
or RNA-like
material (e.g. morplholinos, ribozymes)_
"Polypeptide" refers to a peptide or protein containing two or more amino
acids linked by
peptide bonds, and includes peptides, oligorners, proteins, and the like.
Polypeptides can contain
natural, modified, or synthetic amino acids. Polypeptides can also be modified
naturally, such as
by post-translational processing, or chemically, such as atnidation acylation,
cross-linking,
glycosylation, pegylation, and the like.
The terms "antibody" and "immunoglobulin" are used interchangeably in the
broadest
sense and include monoclonal antibodies (full-length or intact monoclonal
antibodies), polyclonal
antibodies, humanized, multivalent antibodies, multispecific antibodies (e.g.,
bispecific antibodies
so long as they exhibit the desired biological activity), and antibody
fragments.
"Antibody fragnments" contain a portion of an intact antibody, generally the
antigen
binding or variable region of the intact antibody. Examples of antibody
fragments include, but are
not limited to Fab fragments, Fab' fragments, Fd' fragment, Fv fragment, Fd
fragment, F(ab')7
fragment, dAb fragment, hingeless antibodies, single chain antibodies,
diabodies, single arm
antigen binding molecules (containing a light chain, a heavy chain and a N-
terminally truncated
heavy chain constant region sufficient to form a Fc region capable of
increasing the half life of the
single arm antigen binding molecule), and linear antibodies, .
The term "molecular entity" refers to any defined inorganic or organic
molecule that is
either naturally occurring or is produced synthetically. Such molecules
include, but are not limited
to, biomolecules as described above, simple and complex molecules, acids and
alkalis, alcohols,
aldehydes, arenas, amides, amines, esters, ethers, ketones, metals, salts, and
derivatives of any of
the aforementioned molecules.
The term "fi-agment" refers to a portion of a polynucleotide or poiypeptide
sequence that
comprises at least 15 consecutive nucleotides or 5 consecutive amino acid
residues, respectively.
8

CA 02747184 2011-07-22
Furthermore, these "fragments" typically retain the biological activity and/or
some functional
characteristics of the parent polypeptide e.g_ antigenicity or structural
domain characteristics.
The term "prostatic secretory protein 94" or "PSP94" refers to a 94 amino acid
protein
secreted by the prostate that functions as a tumor suppressor. PSI-194 is the
mature protein that is
S amino acid residues Ito 94 of the full-length 114 amino acid protein of SEQ
ID NO:1. The terms
"Prostate Secretory protein 94", "PSP94", "Prostate Secreted Seminal Plasma
Protein", "Seminal
Plasma Beta-Inhibits", "Lmmunoglobulin-binding factor", "1GBF", and "PN44" are
used
interchangeably herein.
The term "PSA" refers to prostate specific antigen. "PSA" is also known as
kallikrein III,
seminin, semenogelasc, y-seminoprotein and P-30 antigen. PSA is a protein
produced by prostate
cells that acts as a serine protease. PSA can be found in seminal fluid and
prostate tissue. This
enzyme can participate in the dissolution of the seminal fluid coagulum and
plays an important
role in fertility. Some PSA can escape the seminal fluid and be found in the
serum. Most PSA in
serum is bound to serum proteins, which is referred to as "bound PSA". PSA
serum levels are
usually elevated in men with prostate cancer. A serum PSA test is currently
the only method used
to screen for early detection of prostate cancer. "Free PSA" refers to PSA
that is not bound to
serum proteins.
The term "total PSA" refers to the sum of free PSA and bound PSA. The terms
"biological
sample" and "test sample" are used interchangeably and refer to all biological
fluids and excretions
isolated from any given subject. Such samples include, but are not limited to,
blood, blood serum,
blood plasma, urine, semen, seminal fluid, seminal plasma, prostatic fluid,
pre-ejaculatory fluid
(Cowper's fluid), excreta, tears, saliva, sweat, biopsy, ascites,
cerebrospinal fluid, lymph, marrow,
hair or tissue extract samples such as homogenized tissue, and cellular
extracts, and combinations
thereof. Tissue samples include samples of tumors.
The term "host cell" refers to a cell that has been transformed or
transfected, or is capable
of transformation or transfection by an exogenous polynticleotide sequence- It
is understood that
such terms refer not only to the particular subject cell but also to the
progeny or potential progeny
of such a cell. Since certain modifications may occur in succeeding
generations due to either
mutation or environmental influences, such progeny may not, in fact, be
identical to the parent
cell, but are still included within the scope of the term as used herein-
The term "specific binding" refers to an interaction between two biomoleeules
that occurs
under specific conditions. The binding of two biotnolecules is considered to
be specific when the
interaction between said molecules is substantial- In the context of the
invention, a binding
reaction is considered substantial when the signal of the peak representing
the biomolecule is at
least twice that of the signal arising from the coincidental detection of non-
biomolecule associated
ions in approximately the same mass range, which is the peak as a signal to
noise ratio of at least
two- Moreover, the phrase "specific conditions" refers to reaction conditions
that permit, enable,
9

CA 02747184 2011-07-22
or facilitate the binding of said molecules such as pH, salt, detergent and
other conditions known
to those skilled in the art
The term "interaction" relates to the direct or indirect binding or alteration
of biological
activity of a biomoleculc.
A reagent may be "immobilized" on or in a support by forming a covalent bond
between a
functional group of the reagent and a reactive group on the surface of the
solid phase- In other
embodiments, the reagent is "immobilized" on the solid phase by adsorption and
ionic binding or
may be entrapped in the solid phase, e_g., within cells or lattice type
polymers or microcapsules
(See Holenberg er al., in Enzymes as Drugs, John Wiley & Sons NY (1981), pages
396-411). The
reagent should essentially retain its ability to bind to and/or modify the
polypeptide of interest
once immobilized to the solid phase.
The term "differential diagnosis" refers to a diagnostic decision between
healthy and
different disease stales, including various stages of a specific disease. A
subject is diagnosed as
healthy or to be suffering from a specific disease, or a specific stage of a
disease based on a set of
hypotheses that allow for the distinction between healthy and one or more
stages of the disease. A
choice between healthy and one or more stages of disease depends on a
significant difference
between each hypothesis. Under the same principle, a "differential diagnosis"
may also refer to a
diagnostic decision between one disease type as compared to another (e-g.,
prostate cancer versus a
non-malignant disease of the prostate).
The term "prostate cancer" refers to a malignant neoplasm of the prostate
within a given
subject, wherein the neoplasm is of epithelial origin and is also referred to
as a carcinoma of the
prostate. Prostate cancer can be defined according to its type, stage and/or
grade. Typical staging
systems include the Jewett-Whitmore system and the TNM system (the system
adopted by the
American Joint Committee on Cancer and the International Union Against
Cancer). A typical
grading system is the Gleason Score which is a measure of tumour
aggressiveness based on
pathological examination of tissue biopsy). The term "prostate cancer", when
used without
qualification, includes both localized and metastasised prostate cancer. The
term "prostate cancer"
can be qualified by the terms "localized" or "metastasised" to differentiate
between different types
of tumour as those words are defined herein. The terms "prostate cancer" and
"malignant disease
of the prostate" are used interchangeably herein.
The tern-is "neoplasm" or "tumour" may be used interchangeably and refer to an
abnormal
mass of tissue wherein the growth of the mass surpasses and is not coordinated
with the growth of
normal tissue- A neoplasm or tumour may be defined as "benign" or "malignant"
depending on
the following characteristics: degree of cellular differentiation including
morphology and
functionality, rate of growth, local invasion and metastasis. A "benign"
neoplasm is generally well
differentiated, has characteristically slower growth than a malignant neoplasm
and remains
localised to the site of origin. In addition a benign neoplasm does not have
the capacity to

CA 02747184 2011-07-22
infiltrate, invade or metastasise to distant sites. A "malignant" neoplasm is
generally poorly
differentiated (anaplasia), has characteristically rapid growth accompanied by
progressive
infiltration, invasion and destruction of the surrounding tissue- Furthermore,
a malignant
neoplasm has to capacity to metastasise to distant sites.
The term "differentiation" refers to the extent to which parenchymal cells
resemble
comparable normal cells both morphologically and functionally.
The term "metastasis" refers to spread or migration of cancerous cells from a
primary
(original) tumour to another organ or tissue, and is typically identifiable by
the presence of a
"secondary tumour" or "secondary cell mass" of the tissue type of the primary
(original) tumour
and not of that of the organ or tissue in which the secondary (metastatic)
tumour is located. For
example, a prostate cancer that has migrated to bone is said to be
metastasised prostate cancer, and
consists of cancerous prostate cancer cells in the prostate as well as
cancerous prostate cancer cells
growing in bone tissue.
The terms "a non-malignant disease of the prostate", "non-prostate cancer
state" and
"benign prostatic disease" may be used interchangeably and refer to a disease
state of the prostate
that has not been classified as prostate cancer according to specific
diagnostic methods including
but not limited to rectal palpitation, PSA scoring, transrectal
ultrasonography and tissue biopsy,
Such diseases include, but are not limited to, an inflammation of prostatic
tissue (i.e., chronic
bacterial prostatitis, acute bacterial prostatitis, chronic abacterial
prostatitis) and benign prostate
hyperplasia_
The term "healthy" refers to an absence of any malignant or non-malignant
disease of the
prostate; thus, a "healthy individual" may have other diseases or conditions
that would normally
not be considered "healthy". A "healthy" individual demonstrates an absence of
any malignant or
non-malignant disease of the prostate.
The term "pre-cancerous lesion of the prostate" or "precancerous prostate
lesion" refers to
a biological change within the prostate such that it becomes susceptible to
the development of a
malignant neoplasm. More specifically, a pre-cancerous lesion of the prostate
is a preliminary
stage of a prostate cancer. Causes of a pre-cancerous lesion may include, but
are not limited to,
genetic predisposition and exposure to cancer-causing agents (carcinogens);
such cancer causing
agents include agents that cause genetic damage and induce neoplastic
transformation of a cell-
The term "neoplastic transformation of a cell" refers to an alteration in
normal cell
physiology and includes, but is not limited to, self-sufficiency in growth
signals, insensitivity to
growth-inhibitory (anti-growth) signals, evasion of programmed cell death,
limitless replicative
potential, sustained angiogenesis, and tissue invasion and metastasis,
The term "differentially present"refers to differences in the quantity of a
biomolecule
present in samples taken from prostate cancer patients as compared to samples
taken from subjects
having a non-malignant disease of the prostate or healthy subjects.
Furthermore, a biomolecule is
Ii

CA 02747184 2011-07-22
differentially present between two samples if the quantity of said biomolecule
in one sample
population is significantly different (defined statistically) from the
quantity of said biomolecule in
another sample population. For example, a given biomolecule may be present at
elevated,
decreased, or absent levels in samples of taken from subjects having prostate
cancer compared to
those taken from subjects who do not have a prostate cancer.
The term "biological activity" may be used interchangeably with the terms
"biologically
active", "bioactivity" or "activity" and, for the purposes herein, means an
effector or antigenic
function that is directly or indirectly performed by a biomarker of the
invention (whether in its
native or denatured conformation), derivative or fragment thereof. Effector
functions include
phosphorylation (kinase activity) or activation of other molecules, induction
of differentiation,
rnitogenie or growth promoting activity, signal transduction, immune
modulation, DNA regulatory
f uictions and the like- Antigenic functions include possession of an epitope
or antigenic site that
is capable of cross-reacting with antibodies raised against a naturally
occurring or denatured
biomarker of the invention, derivative or fragment thereof Accordingly, a
biological activity of
such a protein can be that it functions as regulator of a signalling pathway
of a target cell. Such a
signalling pathway can, fir example, modulate cell differentiation,
proliferation and/or migration
of such a cell, as well as tissue invasion, tumour development and/or
metastasis. A target cell
according to the invention can be a neoplastic or cancer cell.
The terms "neoplastic cell" and "neoplastie tissue" refer to a cell or tissue,
respectively,
that has undergone significant cellular changes (transformation). Such
cellular changes are
manifested by an escape from specific control mechanisms, increased growth
potential, alteration
in the cell surface, karyotypic abnormalities, morphological and biochemical
deviations from the
norm, and other attributes conferring the ability to invade, metastasise and
kill-
The term "diagnostic assay" can be used interchangeably with "diagnostic
method" and
refers to the detection of the presence or nature of a pathologic condition.
Diagnostic assays differ
in their sensitiviry and specificity, and their relative usefulness as a
diagnostic tool can be
measured using ROC-AUC statistics.
Herein, the term "true positives" refers to those subjects having a localized
or a
metastasised cancer of the prostate or a benign prostate disease, a
precancerous prostatic lesion, or
an acute or a chronic inflammation of prostatic tissue and who are categorized
as such by the
diagnostic assay. Depending on context, the term "true positives" may also
refer to those subjects
having either prostate cancer or a non-malignant disease of the prostate, who
are categorized as
such by the diagnostic assay.
Herein, the term "false negatives" refers to those subjects having either a
localized or a
metastasised cancer of the prostate, a benign prostate disease, a precancerous
prostatic lesion, or an
acute or a chronic inflammation of prostatic tissue and who are not
categorised as such by the
diagnostic assay. Depending on context, the tens "false negatives" may also
refer to those subjects
12

CA 02747184 2011-07-22
having either prostate cancer or a non-malignant disease of the prostate and
who are not
categorized as such by the diagnostic assay.
Herein, the term "true negatives" refers to those subjects who do not have a
localized or a
metastasised cancer of the prostate, a benign prostate disease, a precancerous
prostatic lesion, or an
acute or a chronic inflammation of prostatic tissue and who are categorized as
such by the
diagnostic assay. Depending on context, the term "true negatives" may also
refer to those subjects
who do not have prostate cancer or a non-malignant disease of the prostate and
who are
categorized as such by the diagnostic assay.
Herein, the term "false positives" refers to those subjects who do not have a
localized or a
metastasised cancer of the prostate, a benign prostate disease, a precancerous
prostatic lesion, or an
acute or a chronic inflammation of prostatic tissue but are categorized by the
diagnostic assay as
having a localized or metastasised cancer of the prostate, a benign prostate
disease, a precancerous
prostatic lesion or an acute or chronic inflammation of prostatic tissue.
Depending on context, the
term "false positives" may also refer to those subjects who do not have
prostate cancer or a non-
malignant disease of the prostate but. are categorized by the diagnostic assay
as having prostate
cancer or a non-malignant disease of the prostate.
The term "sensitivity", as used herein in the context of its application to
diagnostic assays,
refers to the proportion of all subjects with localized or metastasised cancer
of the prostate, a
benign prostate disease, a precancerous prostatic lesion, or an acute or a
chronic inflammation of
prostatic tissue that are correctly identified as such (that is, the number of
true positives divided by
the sum of the number of true positives and false negatives).
The term "specificity" of a diagnostic assay, as used herein in the context of
its application
to diagnostic assays, refers to the proportion of all subjects with neither
localized or metastasised
cancer of the prostate nor a benign prostate disease, a precancerous prostatie
lesion, or an acute or
a chronic inflammation of prostatic tissue that are correctly identified as
such (that is, the number
of true negatives divided by the sum of the number of true negatives and false
positives).
The term "adsorbent" refers to any material that is capable of accumulating
(binding) a
given biomolecule. The adsorbent typically coats a biologically active surface
and is composed of
a single material or a plurality of different materials that are capable of
binding a biomolecule_
Such materials include, but are not limited to, anion exchange materials,
cation exchange
materials, metal chelators, polynucleotides, oligonucleotides, peptides,
antibodies, naturally
occurring compounds, synthetic compounds, etc.
The phrase "biologically active surface" refers to any two- or three-
dimensional extensions
of a material that biomolecules can bind to, or interact with, due to the
specific biochemical
properties of this material and those of the biomolecules. Such biochemical
properties include, but
are not limited to, ionic character (charge), hydrophobicity, or
hydrophilicity.
13

CA 02747184 2011-07-22
The phrase "binding biomolecule" refers to a molecule that displays an
affinity for another
biomolecule-
The term "immunogen" may be used interchangeably with the phrase "immunising
agent"
and refers to any substance or organism that provokes an immune response when
introduced into
the body of a given subject- All immunogens are considered as antigens and, in
the context of the
invention, can be defined on the basis of their immunogenicity, wherein
"immtmogenicity" refers
to the ability of the immunogen to induce either a humoral or a cell-mediated
immune response. In
the context of the invention an immunogen that induces a "humoral -immune
response" activates
antibody production and secretion by cells of the B-lymphocyte lineage (B-
cells) and thus can be
used to for antibody production as described herein. Such immunogens may be
polysaccharides,
proteins, lipids or nucleic acids, or they may be lipids or nucleic acids that
are eomplexed to either
a polysaccharide or a protein.
The term "solution" refers to a homogeneous mixture of two or more substances-
Solutions may include, but are not limited to buffers, substrate solutions,
elution solutions, wash
solutions, detection solutions, standardisation solutions, chemical solutions,
solvents, etc-
The phrase "coupling buffer" refers to a solution that is used to promote
covalent binding
of biomolecules to a biological surface.
The phrase "blocking buffer" refers to a solution that is used to (prevent)
block unbound
binding sites of a given biological surface from interacting with biomolecules
in an unspecific
manner.
The term "chromatography" refers to any method of separating biomolecules
within a
given sample such that the original native state of a given biomolecule is
retained.. Separation of a
biomoleculc from other biomolecules within a given sample for the purpose of
etuiclunent,
purification and/or analysis, may be achieved by methods including, but not
limited to, size
exclusion chromatography, ion exchange chromatography, hydrophobic and
hydrophilic
interaction chromatography, metal affinity chromatography, wherein "metal"
refers to metal ions
(e.g. nickel, copper, gallium, zinc, iron or cobalt) of all chemically
possible valences, or ligand
affinity chromatography wherein "ligand" refers to binding molecules,
preferably proteins,
antibodies, or DNA_ Generally, chromatography uses biologically active
surfaces as adsorbents to
selectively accumulate certain biomolecules.
The phrase "mass spectrometry" refers to a method comprising employing an
ionisation
source to generate gas phase ions from a biological entity of a sample
presented on a biologically
active surface, and detecting the gas phase ions with an ion detector.
Comparison of the time the
gas phase ions take to reach the ion detector from the moment of ionisation
with a calibration
equation derived from at least one molecule of known mass allows the
calculation of the estimated
mass to charge ratio of the ion being detected.
14

CA 02747184 2011-07-22
The phrases "mass to charge ratio", "m/z ratio" or "in/,." can be used
interchangeably and
refer to the ratio of the molecular weight (grams per mole) of an ion detected
by mass
spectrometry to the number of charges the ion carries- Thus a single
biomolecule can be assigned
more than One mass to charge ratio by a mass spectrometer if that biomolecule
can be ionised into
more than one species each of which carries a different number of charges.
The terms "detect", "detection" or "detecting" refer to the identification of
the presence,
absence, or quantity of a given biomolecule.
The acronym "ROC-AUC" refers to the area under a receiver operator
characteristic curve.
This is a widely accepted measure of diagnostic utility of some tool, taking
into account both the
sensitivity and specificity of the tool. Typically, ROC-AUC ranges from 0.5 to
1.0, where a value
of 0.5 indicates the tool has no diagnostic value and a value of 1.0 indicates
the tool has 100%
sensitivity and 100% specificity,
The term "hypertension" refers to elevated blood pressure. A definition of
hypertension is
arbitrary because the risk of cardiovascular disease related to blood pressure
level increases
steadily across the spectrum of blood pressure values, however optimum blood
pressure is defined
as less than 115/75 mm Hg_ Normal blood pressure (the level associated with
minimal risk) for
adults 18 years of age or older is a systolic blood pressure of less than 120
mm Hg and a diastolic
blood pressure of less than 80 mm l-Ig_ For the general population,
hypertension is defined as a
systolic BP of 140 mm Hg or higher or a diastolic BP of 90 mm Hg or higher.
(Lewington S,
Clarke R,, Qizilbash N, et al: Age-specific relevance of usual blood pressure
to vascular mortality-
a meta-analysis of individual data from one million adults in 61 prospective
studies. Lancet
360:1903, 2002)_ The term "sensitivity" refers to the proportion of patients
with the outcome in
whom the results of the decision rule are abnormal. Typically, the outcome is
disadvantageous to
the patient. The term "specificity" refers to the proportion of patients
without the outcome in
whom the results of the decision Wile are normal.
It is to be understood that the present invention is not limited to the
particular materials
and methods described or equipment, as these may vary. It is also to be
understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not
intended to limit the scope of the present invention, which will be limited
only by the appended
claims.
it should be noted that as used herein and in the appended claims, the
singular forms "a,"
"an," and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for
example, a reference to "an antibody" is a reference to one or more antibodies
and derivatives
thereof known to those skilled in the art, and so forth.

CA 02747184 2011-07-22
PSP94
PSP94 is a versatile protein that plays a role in several biological processes
within the
reproductive tract ranging from modulating the circulation of follicle-
stimulating hormone (FSH)
to inducing apoptosis in prostate cancer cells (Sheth at a1., 1984; Chao et
al., 1996; Hirano at at.,
1996; Garde at al., 1999; Shukeir at al., 2003). It is one of the three major
proteins secreted by the
normal human prostate gland. As a secreted protein, this molecule is found in
a variety of bodily
fluids including serum (Teni et al., 1988; Reeves et al., 2005; van Huizen at
al., 2005), urine (Teni
et al., 1988; Liu et al., 1993), seminal plasma fluid (Sheth et al., 1984;
Dube et al., 1987; von der
Kammer at aL, 1991) and mucous gland secretions (Weiber at at., 1990). PSP94
occurs in both the
free and bound forms in serum (Wu at al., 1999).
full-length PSP94 has the following sequence:
MN VLLGSVVIFATFVTLCNASCYFIPNECiVPGDSTRKCMDLKGNKHPiNS
EWQ'fDNCETCTCYETETSCCTLV STPV GYDK DNCQRIFKKEDCKY!V VEK
KDPKKTCSVSEWTT (SEQ ID NO: 1; Accession No. AB29732.1/G1:460569)
Mature PSP94 has the following sequence:
S CYFIPNEG VPGDSTRKCMDLKGNKHPTNSE W QTDNC ETCTC Y ETFI SCC
TLVSTPVGYDK.DNCQRiFKKEDCKYTVV.EKKDPKKTCSVSEWIF (SEQ ID
NO:2)
Dia-enustic Toole
Although PSP94 has been shown to be a useful discriminatory factor for
diagnosis and/or
prognosis of prostate cancer, diagnostic tools utilizing this protein are both
invasive and lacking
sensitivity. A diagnostic tool utilising PSP94 and total PSA has not yet been
described. This
improves the discrimiaiatory value for prostate cancer over each of the
markers when used alone.
In addition to this, urine samples are the preferred samples for diagnostic
tools described herein,
making the test ideal for clinical application. Embodiments of the invention
are non-invasive and
cost-effective.
The present invention relates to methods for differential diagnosis of
prostate cancer or a
non-malignant disease of the prostate by detecting PSP94 and total PSA, within
a biological
sample of a given subject, comparing results with samples from healthy
subjects, subjects having a
non-malignant disease of the prostate and subjects having prostate cancer,
wherein the comparison
allows for the differential diagnosis of a subject as healthy, having non-
malignant disease of the
prostate or having prostate cancer.
One aspect of the invention includes a method for diagnosing prostate cancer
in a subject
comprising; (a) detecting a quantity, presence or absence of PSP94 and total
PSA; and (b)
classifying the subject as having or not having prostate cancer.
16

CA 02747184 2011-07-22
In an embodiment of the invention, the step of classifying the subject
comprises
comparing the quantity, presence or absence of PSP94 and total PSA, with a
reference value
indicative of a prostate cancer. The reference value comprises PSP94 and total
PSA. A further
aspect of the invention includes a method for differential diagnosis of
prostate cancer and non-
malignant disease of the prostate in a subject, comprising: (a) detecting a
quantity, presence or
absence of PSP94 and total PSA, in a biological sample; and (b) classifying
the subject as having
prostate cancer, non-malignant disease of the prostate, or as healthy, based
on the quantity,
presence or absence of PSP94 and total PSA..
In an embodiment of the invention, the step of classifying the subject
comprises
comparing the quantity, presence or absence of PSP94 and total PSA, with a
reference value
indicative of prostate cancer and a reference value indicative of a non-
malignant disease of the
prostate. Reference values comprise PSP94 and total PSA, characterised as
being diagnostic for
prostate cancer or for a non-malignant disease of the prostate.
A further aspect of the invention includes a method for differential diagnosis
of healthy,
non-malignant disease of the prostate, precancerous prostatic lesion,
localized cancer of the
prostate, metastasised cancer of the prostate, and acute or chronic
inflammation of prostatic tissue
in a subject, comprising: (a) detecting a quantity, presence or absence of
PSP94 and total PSA, in a
biological sample; and (b) classifying the subject as having non-malignant
disease of the prostate,
preeancerous prostate lesion, localized cancer of the prostate, metastasised
cancer of the prostate,
and/or acute or chronic inflammation of prostatic tissue, or as healthy, based
on the quantity,
presence or absence of PSP94 and total PSA, in the biological sample.
Reference values may
include values for good health, non-malignant disease of the prostate,
precancerous prostate lesion,
localized cancer of the prostate, metastasised cancer of the prostate, and/or
acute or chronic
inflammation of prostatic tissue-
In one embodiment, a biologically active surface comprises an adsorbent
comprising
silicon dioxide molecules. in another embodiment, a biologically active
surface comprises an
adsorbent comprised of antibodies. Antibodies may be antibodies specific to
PSP94 and PSA.
Biologically active surfaces useful for practicing the methods of the
invention are further described
in greater detail below,
In addition, other methods of determining a quantity, presence or absence of
PSP94 and
total PSA, in a biological sample can be utilized, such as ELISA utilizing
antibodies targeted to a
biornarker of the invention. In any of the embodiments of the methods
described above, PSP94 and
total PSA, may be detected within a given biological sample. Detection of
biomolecules of the
invention is based on specific sample pre-treatment conditions, the p14 of
binding conditions, the
adsorbent used on the biologically active surface. In one embodiment of the
invention, a
biomolecule of the invention can include PSP94 and total PSA, and may be used
to diagnose a
subject as being healthy, having a non-malignant disease of the prostate,
having a precancerous
17

CA 02747184 2011-07-22
prostatic lesion, having a localized cancer of the prostate, having a
metastasised cancer of the
prostate, or having an acute or a chronic inflammation of prostatic tissue. In
another embodiment
of the invention, PSP94 and total PSA may be used in combination with at least
one other
biomolecule to diagnose a subject as being healthy, having of a non-malignant
disease of the
prostate, having a precancerous prostatic lesion, having a localized cancer of
the prostate, having a
metastasised cancer of the prostate, or having an acute or a chronic
inflammation of prostatic
tissue-
In yet another embodiment of the invention, detection and/or quantification of
biomolecules, including PSP94 and total PSA, may be used in combination with
another
diagnostic tool to diagnose a subject as being healthy, having a non-malignant
disease of the
prostate, having a precancerous prostatic lesion, having a localized cancer of
the prostate, having a
metastasised cancer of the prostate, or having an acute or a chronic
inflammation of prostatic
tissue. For example, PSP94 and total PSA may be used in combination with at
least one other
diagnostic tool specific for prostate cancer detection such as, but not
limited to, DRE, rectal
palpitation, biopsy evaluation using Gleason scoring, radiography, proteo-
imaging, and
symptomological evaluation by a qualified clinician. In another embodiment,
PSP94 and total
PSA, may also be combined with the detection of PCA3 (also known as DD3)
and/or a prostate
disease specific microRNA pattern-
Methods for detecting biomolecules according to the invention have many
applications.
For example, PSP94 and total PSA can be measured to differentiate between
healthy subjects,
subjects having a non-malignant disease of the prostate, subjects having a
precanccrous prostatic
lesion, subjects having a localized cancer of the prostate, subjects having a
metastasised cancer of
the prostate, or subjects with an acute or a chronic inflammation of prostatic
tissue, and thus are
useful as an aid in diagnosis of a non-malignanr disease of the prostate, a
precancerous prostatic
lesion, or a localized cancer of the prostate, a metastasised cancer of the
prostate, or an acute or a
chronic inflammation of prostatic tissue. Alternatively, said biomolecules may
be used to
diagnose a subject as being healthy.
Another aspect of the invention includes an in vitro method for diagnosis of
prostate
cancer in a subject comprising detecting differentially expressed biomarkers
in a biological sample
by, (a) contacting a sample with a binding molecule specific for PSP94 and a
second binding
molecule specific for PSA, and (b) detecting a quantity, presence or absence
of PSP94 and total
PSA, wherein the quantity, presence, or absence of PSP94 and total PSA, and
(c) diagnosing the
subject as healthy or having prostate cancer,
A further aspect of the invention includes a method for in vitro differential
diagnosis of
prostate cancer and non-malignant disease of the prostate in a subject,
comprising detecting one or
more differentially expressed biomarkers in a biological sample: (a)
contacting a sample with a
first binding molecule specific for PSP94 and a second binding molecule
specific for PSA, (b)
1$

CA 02747184 2011-07-22
detecting a quantity, presence or absence of PSP94 and total PSA in the
sample, and (c) diagnosing
the subject as having prostate cancer, and/or having a non-malignant disease
of the prostate, or as
being healthy.
Still a further aspect of the invention includes an in virro method for
differential diagnosis
of healthy, prostate cancer, non-malignant disease of the prostate,
precancerous prostatic lesion,
localized cancer of the prostate, metastasised cancer of the prostate, and
acute or chronic
inflammation of prostatic tissue in a subject, comprising detection of PSP94
and total PSA, in a
biological sample by; (a) contacting a sample with a first binding molecule
specific for PSP94 and
a second binding molecule specific for PSA, and (b) detecting a quantity,
presence or absence of
PSP94 and total PSA, wherein the presence or absence of PSP94 and total PSA,
allows for the
differential diagnosis of the subject as healthy, having non-malignant disease
of the prostate, .
precancerous prostate lesions, localized cancer of the prostate, metastasised
cancer of the prostate,
and/or having acute or chronic inflammation of the prostate, or as being
healthy-
Binding molecules include, but are not limited to polynucleotides,
polypeptides (e.g.,
=monoclonal and/or polyclonal antibodies, antigens, etc.), carbohydrates
(e.g., sugars), fatty acids,
lipids, steroids, or combinations thereof (e.g. glycoproteins,
ribonucleoproteir)s, lipoproteins),
compounds or synthetic molecules. In one preferred embodiment, binding
molecules are
antibodies specific for PS1394 or PSA_ For example, antibodies or fragments
thercof may be
utilized for the detection of PSP94 and PSA. Depending on the nature of a
biological sample, it is
possible to determine not only presence of a biomolecule, but also its
cellular distribution. For
example, in a blood serum sample, only serum levels Of a given biomolecule can
be detected,
whereas its level of expression and cellular localisation can be detected in
histological samples.
In another example, an antibody directed against a biomolecule of the
invention that is
coupled to an enzyme can be detected using a chromogenic substrate that can be
recognized and
cleaved by the enzyme to produce a chemical moiety, which is readily detected
using
spectrometric, fluorirnetric or visual means- Enzymes used for labelling
include, but are not
limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast
alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate
isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase,
ribonuclease, unease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase and
acetylcholinesterase. Detection may also be accomplished by visual comparison
of the extent of
the enzymatic reaction of a substrate with that of similarly prepared
standards. Alternatively,
radio-labelled antibodies can be detected using a gamma or a scintillation
counter, or they can be
detected using autoradiography. In another example, fluorescently labelled
antibodies are detected
based on the level at which the attached compound fluoresces following
exposure 10 a given
wavelength- Fluorescent compounds typically used in antibody labelling
include, but are not
limited to, fluorescein isothiocynate (FiTC), rhodamine, phycoerthyrin,
phycocyanin,
19

CA 02747184 2011-07-22
allophycocyani, o-phthaldehyde and fluorescamine. In yet another example,
antibodies coupled to
a chemi- or bioluminescent compound can be detected by determining the
presence of
luminescence. Such compounds include, but are not limited to, lumina],
isoluminal, theromatic
acridinium ester, imidazole, acridinium salt, oxalate ester, luciferin,
luciferase and aetluorin.
Furthermore, in vivo techniques for detecting a biomolecule include
introducing into a
subject a labelled antibody directed against a biomolecule, which can be PSP94
and/or PSA_
In addition, methods of the invention for differential diagnosis of healthy
subjects, subjects
having a non-malignant disease of the prostate, subjects having a precancerous
prostatic lesion,
subjects having a localized cancer of the prostate, subjects having a
metastasised cancer of the
prostate and/or subjects having an acute or chronic inflammation of prostatic
tissue, described
herein may be combined with other diagnostic methods to improve the outcome of
the differential
diagnosis.
Methods of the invention can also be used for differential diagnosis of
healthy subjects,
subjects having a precancerous prostatic lesions, subjects having a non-
malignant disease of the
prostate, subjects having a localized cancer of the prostate, subjects having
metastasised cancer of
the prostate, and/or subjects laving acute or chronic inflammation of the
prostate, or any two or
more of the above states.
Biological samples of the invention
Typically, PSP94 and PSA are detected in urine samples, but their detection is
not limited
to urine samples. Biomolecules of the invention can be detected in blood,
blood serwn, blood
plasma, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-
ejaculatory fluid
(Cowper's fluid), excreta, tears, saliva, sweat, biopsy, ascites,
cerebrospinal fluid, lymph, or tissue
extract (biopsy) samples. Preferably, biological samples used to detect
biomolecules of the
invention are urine, semen, seminal fluid, seminal plasma, prostatic fluid, or
pre-ejaculatory fluid
(Cowper's fluid). Furthermore, biological samples can be isolated from
mammalian subjects,
preferably humans.
A subject that is said to have a prostate cancer possesses morphological,
biochemical and
functional alterations of their prostatic tissue such that the tissue can be
characterised as a
malignant neoplasm. The stage to which a prostate cancer has progressed can be
determined using
known methods currently available to those skilled in the art (e.g., Union
Internationale Contre
Cancer (UICC) system or American Joint Committee on Cancer (AJC))_ Currently,
the most
widely used method for determining the extent of malignancy of a prostatic
neoplasm is the
Gleason Grading system- Gleason grading is based exclusively on the
architectural pattern of the
glands of it prostatic neoplasm, wherein the ability of neoplastic cells to
structure themselves into
glands resembling those of the normal prostate is evaluated using a scale of i
to 5. For example,
neoplastic cells that are able to architecturally structure themselves such
that they resemble normal

CA 02747184 2011-07-22
prostate gland structure are graded 1-2, whereas neoplastic cells that are
unable to do so are graded
4-5. As known to those skilled in the art, a prostatic neoplasm whose tumour
structure is nearly
normal will tend to behave, biologically, as normal tissue and therefore it is
unlikely that it will be
aggressively malignanT_ Gleason score may be integrated with other grading
methods and/or
staging systems to determine cancer stage.
A subject is said to have a non-malignant disease of the prostate possesses
morphological
and/or biochemical alterations of their prostatic tissue but does not exhibit
malignant neoplastic
properties known to those skilled in the art. Such diseases include, but are
not limited to,
inflammatory and proliferative lesions, as well as benign disorders of the
prostate. Within the
context of the invention, inflammatory lesions encompass acute and chronic
bacterial prostatitis, as
well as chronic abactorial prostatitis, proliferative lesions include benign
prostate hyperplasia
(BPI I).
Biologically active Surfaces
Biologically active surfaces include, but are not limited to, surfaces that
contain adsorbents
with anion exchange properties (adsorbents that are positively charged),
cation exchange
properties (adsorbents that are negatively charged), hydrophobic properties,
reverse phase
chemistry, groups such as nitriloacetic acid that immobilize metal ions such
as nickel, gallium,
copper, or zinc (metal affinity interaction), or biomoleeules such as
proteins, antibodies, nucleic
acids, or protein binding sequences, eovalently bound to the surface via
carboiiyl diimida7 le
moieties or epoxy groups (specific affinity interaction).
These surfaces may be located on matrices like polysaccharides such as
sepharose, e.g.
anion exchange surfaces or hydrophobic interaction surfaces, or solid metals,
e.g_ antibodies
coupled to magnetic beads or a metal surface. Surfaces may also include gold-
plated surfaces such
ac those used for BiacoreTM Sensor Chip technology.
Biologically active surfaces are able to adsorb biomolecuJes like nucleotides,
polynuclcotides, amino acids, polypeptides (e.g., monoclonal and/or polyclonal
antibodies),
steroids, carbohydrates (e-g., sugars), fatty acids, lipids, hormones, and
combinations thereof(e.g.,
glycoproteins, ribonucleoproteins, lipoproteins).
Devices that use biologically active surfaces to selectively adsorb
biomolecules can be
chromatography columns for Fast Protein Liquid Chromatography (FPLC) and High
Pressure
Liquid Chromatography (HPLC), where the matrix, e.g. a polysaccharide,
carrying the biologically
active surface, is filled into vessels (usually referred to as "columns") made
of glass, steel, or
synthetic materials like polyetheretherketone (PEEK).
In yet another embodiment, devices that use biologically active surfaces to
selectively
adsorb biomolecules may be metal strips carrying thin layers of a biologically
active surface on
one or more spots of the strip surface to be used as probes for gas phase ion
spectrometry analysis,
21

CA 02747184 2011-07-22
for example the PS20 ProteinChip0 array for (Ciphergen Biosystems, Inc.) for
surface-enhanced
laser desorption/ionization (SELDI) analysis.
Detection of biomolecnles of the invention
In another aspect of the invention, biomolecules (e.g., PSP94 and total PSA)
can be
detected using other known methods. For example, an in vitro binding assay can
be used to detect
a. biomolecule within a biological sample of a given subject. A given
biomolecule can be detected
within a biological sample by contacting the biological sample from a given
subject with specific
binding molecule(s) under conditions conducive for an interaction between the
given binding
molecule(s) and a biomolecule. Binding molecules include, but are not limited
to, nucleic acids,
nucleotides, polynucleotides, amino acids, polypeptides (e.g., monoclonal
and/or polyclonal
antibodies, and antigens), carbohydrates (e.g., sugars), fatty acids, lipids,
steroids, or combinations
thereof (e.g. glycoproteins, ribonucleoproteins, lipoproteins), compounds or
synthetic molecules.
Preferably, binding molecules are antibodies specific for PSP94 or PSA.
Biomolecules detected
using the above-mentioned binding molecules include, but are not limited to,
molecules
comprising nucleic acids, nucleotides, polynucleotides, amino acids,
polypeptides (e.g.,
monoclonal and/or polyclonal antibodies, antigens), carbohydrates (e.g.,
sugars), fatty acids, lipids,
steroids, and combinations thereof (e.g., glycoproteins, ribonucleoproteins,
lipoproteins).
Sandwich Assay
Sandwich assays for detecting a biomolecule, which can be PSP94 and/or PSA,
can be used as a
diagnostic tool for diagnosis of a subject as being healthy, having a non-
malignant disease of the
prostate, having a precancerous prostatie lesion, having a localized cancer of
the prostate, or a
metastasised cancer of the prostate, or having an acute or a chronic
inflammation of prostatic
tissue. Jn the context of the invention, sandwich assays comprise attaching a
monoclonal antibody
to a solid surface such as a plate, tube, bead, or particle, wherein the
antibody is preferably
attached to the well surface of a 96-well microtitre plate- A pre-determined
volume of sample
(e.g., serum, urine, tissue cytosol) containing a subject biomarker is added
to the solid phase
antibody, and the sample is incubated for a period of time at a pre-determined
temperature
conducive for specific binding of subject biomarkers within the given sample
to the solid phase
antibody. Following incubation, the sample fluid is discarded, and the solid
phase is washed with
buffer to remove any unbound material. A second monoclonal antibody (to a
different determinant
on the subject biomarker) is added to the solid phase. This antibody is
labelled with a detector
molecule or atom (c-g-, enzyme, fluorophore, chromophore, or 125I), and the
solid phase is
incubated with the second antibody. The second antibody is decanted and the
solid phase is
22

CA 02747184 2011-07-22
washed with buffer to remove unbound material.
The amount of bound label, which is proportional to the amount of subject
biomarker
present in the sample, can be quantitated.
Kits
A further aspect of the invention comprises a kit for diagnosing a prostate
disease within a
subject comprising: a biologically active surface comprising an adsorbent,
binding solutions, and
instructions to use the kit, wherein the instructions outline a method for
diagnosis of a prostate
cancer in a subject or a method for differential diagnosis of healthy, non-
malignant disease of the
prostate, precancerous prostatic lesion, localized cancer of the prostate,
metastasised cancer of the
prostate, and acute or chronic inflammation of prostatic tissue in a subject
according to the
invention-
Any of the biologically active surfaces described herein may be used to
practice the
invention. In an embodiment of the invention, a biologically active surface
may comprise an
adsorbent comprising of silicon dioxide molecules- In another embodiment of
the invention, a
biologically active surface may comprise an adsorbent comprising antibodies
specific to PSP94
and total PSA_
A further aspect of the invention comprises a kit for diagnosing prostate
disease within a
subject comprising a binding solution, a binding molecule, a detection
substrate, and instructions,
wherein the instructions describe an in vitro method for diagnosis of a
prostate cancer in a subject,
an in vitro method for differential diagnosis of prostate cancer and non-
malignant disease of the
prostate in a subject, or an in vitro method for differential diagnosis of
healthy, prostate cancer,
non-malignant disease of the prostate, precancerous prostatic lesion,
localized cancer of the
prostate, metastasised cancer of the prostate, and acute or chronic
inflammation of prostatic tissue
in a subject.
Yet another aspect of the invention comprises kits using methods of the
invention as
described in another section for differential diagnosis of prostate cancer or
a non-malignant disease
of the prostate, wherein the kits are used to detect biomolecules, which can
be PSP94 and total
PSA. -
Methods used to detect biomolecules, which can be PSP94 and total PSA can also
be used
to determine whether a subject is at risk of developing prostate cancer or has
developed prostate
cancer. Such methods may also be employed in the form of a diagnostic kit
comprising a binding
molecule specific to a biomolecule, which can be PSP94 and PSA, solutions and
materials
necessary for the detection of a biomolecule of the invention, and
instructions to use the kit based
on the above-mentioned methods.
For example, a kit can be used to detect biomolecules such as PSP94 and PSA
and have
many applications. For example, kits can be used to differentiate whether a
subject is healthy, has
23

CA 02747184 2011-07-22
a non-malignant disease of the prostate, or a prostate cancer, thus aiding
diagnosis of a prostate
cancer and/or a non-malignant disease of the prostate. Moreover, kits can be
used to differentiate
whether a subject is healthy, having a non-malignant disease of the prostate,
has a precancerous
prostatie lesion, has a localized cancer of the prostate, has a metastasised
cancer of the prostate, or
has an acute or a chronic inflammation of the prostate.
In an embodiment, a kit may comprise instructions on how to use the kit, a
biologically
active surface comprising an adsorbent, wherein the adsorbent is suitable for
binding one or more
biomolecules of the invention, a denaturation solution for the pre-treatment
of a sample, a binding
solution, and one or more washing solution(s) or instructions for making a
denaturation solution,
binding solution, or washing solution(s), wherein the combination of solutions
allows for the
detection of a biomolecule using gas phase ion spectrometry Stich kits can be
prepared from
materials described in other previously detailed sections (e. g., denaturation
buffer, binding buffer,
adsorbents, washing solution(s), etc.).
In another embodiment, a kit may comprise a first substrate comprising an
adsorbent
thereon (e. g-, a particle functionalised with an adsorbent) and a second
substrate onto which the
first substrate can be positioned to form a probe, which is removably
insertable into a gas phase
ion spectrometer. in other embodiments, a kit may comprise a single substrate,
which is in the
form of a removably insertable probe with adsorbents on the substrate.
In another embodiment, a kit may comprise a binding molecule(s) that
specifically binds
to a biomolecule, which can be PSP94 and/or PSA, a detection reagent,
appropriate solutions and
instructions on how to use the kit. Such kits can be prepared from materials
described above and
known materials. A binding molecule used within such a kit may include, but is
not limited to,
nucleic acids, nucleotides, polynucleotides, amino acids, polypeptides (e.g.,
monoclonal and/or
polyclonal antibodies), carbohydrates (e-g-, sugars), fatty acids, lipids,
steroids, hormones, or a
combination thereof (e.g. glycoproteins, ribonucleoproteins, lipoproteins),
compounds or synthetic
molecules- In another embodiment, a kit comprises a binding molecule or panel
of binding
molecules that specifically bind to PSP94 and/or PSA, a detection reagent,
appropriate solutions
and instructions on how to use the kit. Each binding molecule would be
distinguishable from
every other binding molecule in a panel of binding molecules, yielding easily
interpreted signal for
each of the biomolecules detected by the kit Such kits can be prepared from
the materials
described above and known materials. A binding molecule can include, but is
not limited to,
nucleic acids, nucleotides, polynucleotides, amino acids, polypeptides (e.g.,
monoclonal and/or
polyclonal antibodies), carbohydrates (c-g., sugars), fatty acids, lipids,
steroids, hormones, or a
combination thereof (e-g. glycoproteins, ribonucleoproteins, lipoproteins),
compounds or synthetic
molecules-
1n any of the embodiments described above, a kit may optionally further
comprise a
standard or control biomolecule so that the biomolecules detected within the
biological sample can
24

CA 02747184 2011-07-22
be compared with said standard to determine if the test amount of a marker
detected in a sample is
a diagnostic amount consistent with a diagnosis of a non-malignant disease of
the prostate, a
precancerous prostatic lesion, localized cancer of the prostate, metastasised
cancer of the prostate,
acute or a chronic inflammation of the prostate. Likewise, a biological sample
can be compared
with said standard to determine if the test amount of a marker detected is
said sample is a
diagnostic amount consistent with a diagnosis as healthy.
Patients with hypertension can have higher PSP94 values, even in the absence
of non-
malignant prostate disease or prostate cancer. In any of the embodiments
described above, a
method of diagnosis includes methods where a subject with hypertension is
excluded from said
method- Additionally, any database entries of the described embodiments and/or
reference values
can be obtained from a population of subjects, wherein the population of
subjects excludes
subjects with hypertension-
Exemplaury Methods and Systerrrs for Performing Same
I5 Figure 1 is a flow chart illustrating an example method 100 of diagnosing a
subject. In an
example embodiment, method 100 includes operations 102, 104, 106, 108, 110,
and 112-
Operation 1 02 is performed to determine whether the subject has hypertension.
If so,
method 100 ends. If not, operation 100 proceeds to operation 104.
In an embodiment, operation 102 includes entering a value to correspond that a
subject has
been diagnosed with hypertension by a physician, to some example embodiments,
operation 102
includes measuring the subject's blood pressure, or otherwise determining the
subject's blood
pressure (such as by retrieving it from the patient's medical record or
receiving it from a blood
pressure measurement device)- In some embodiments, the blood pressure is
measured using a
blood pressure measurement device, such as a sphygmomanometer- In some
embodiments the
blood pressure measurement device includes an inflatable cuff attached to a
mercury or aneroid
manometer. In another embodiment, the blood pressure measurement device is an
ambulatory
blood pressure monitor. In some embodiments, the blood pressure measurement
device is a digital
device, such as including a processing device and a memory device. Some
embodiments further
include a data communication device configured to communicate digital data to
another computing
device. Some embodiments include at least one processing device and at least
one memory
device- Some embodiments include programmable electronics including at least
one processing
and at least one memory device,
Once the blood pressure has been measured, operation 102 determines whether
the subject
has hypertension, such as by comparing the blood pressure to a threshold
value. In sonic
embodiments, the subject's age is also considered- A lookup table stored in a
memory device is
used in some embodiments to determine whether the subject has hypertension
based at least in part

CA 02747184 2011-07-22
on the measured blood pressure, and in some embodiments, other factors, such
as the subject's
age
In operation 104, a subject biological sample is received- An example of a
biological
sample is a urine sample. Examples of possible urine samples include spot
collection urine
samples and 24 hour collection urine samples. In another embodiment, a
biological sample is a
blood sample, such as including blood serum. In some embodiments, multiple
biological samples
are used, such as a urine sample and a blood sample. Other embodiments include
other biological
samples or combinations of biological samples, such as those discussed herein
or other biological
samples. The biological samples can be at least temporarily stored in a
container (e.g., vial,
syringe, cup, etc,)_
Following operation 104, the subject biological sample is evaluated through
operations
106 and 108, which can be performed in any sequence or simultaneously. in
operation 106, the
subject biological sample is evaluated to determine a first characteristic of
the subject. An
example of a first characteristic is a PSA value. In operation 108, the
subject biological sample is
evaluated to determine a second characteristic of the subject. An example of a
second
characteristic is a PSP94 value.
Once the first and second characteristics of the subject have been determined,
operation
1 10 is performed to compute a standard score based at least in part on the
first and second
characteristics of the subject- An example of operation 110 is illustrated and
described herein with
reference to Figure 2.
in some embodiments, once the standard score has been computed, operation 112
is
performed to diagnose the subject using the standard score. For example, the
standard score is
compared to two or more possible ranges of scores to determine which of the
ranges the standard
score falls within. Once that range has been identified, the diagnosis is
determined by identifying
a diagnosis associated with the identified range of scores. In some
embodiments, one or more
cutoff values are used to identify boundaries of the ranges- For example, if
the standard score is
less than the cutoff value, the diagnosis is negative, while if the standard
score is greater than or
equal to the cutoff value, the diagnosis is positive.
Operation 112 is not performed in all embodiments. For example, some
embodiments are
a method of generating a standard score. In some embodiments, the standard
score is computed in
operation 110 and is then saved to a memory device- In some embodiments the
standard score is
displayed on a display device. Jn other embodiments, the standard score is
communicated to a
computing device, such as across a network. In some embodiments the standard
score is stored in
the subject's medical record in a medical records database. In yet further
embodiments, the
standard score is stored in a database where it is compiled with the standard
scores of other
subjects for further data processing and analysis-
26

CA 02747184 2011-07-22
Figure 2 is a flow chart illustrating an example method of computing a
standard score
based at least in part on first and second characteristics of a subject, as
illustrated and described
herein as operation 110, shown in Figure 1. However, in some embodiments the
method shown in
Figure 2 is performed independent from method 100, shown in Figure 1. In some
embodiments,
operation 110 includes operations 122, 124, and 126.
Operations 122 and 124 are first performed to receive inputs identifying first
and second
characteristics of the subject. In some embodiments the inputs are provided by
a human, such as a
caregiver or the subject, through a user input device. Examples of user input
devices include a
keyboard, a mouse, a voice recognition system, a touch display, and other user
input devices. A
computing device operates to prompt the user For the inputs in some
embodiments.
In yet an embodiment, the inputs are retrieved from a memory device. An
example of a
memory device is a. database. An example of a database is a medical record of
the subject.
The inputs identifying the first and second characteristics are typically
stored in one or
more memory devices after they have been received. In some embodiments the
inputs are
converted to a standard format prior to storing in the memory device.
Operations 122 and 124 can
be performed in any order, or simultaneously.
Once the inputs have been received, operation 126 performs the computation of
a standard
score using the first and second characteristics of the subject that were
input. In some
embodiments, the computation utilizes a predetermined mathematical formula.
The mathematical
formula is determined, for example, by using logistic regression to fit a set
of data to a logit
function logistic curve. The set of data is, for example, a compilation of
subject data identifying
values of first and second characteristics of patients. In one example
embodiment, the data is
normalized using Iog(PSA) and log(PSP94) before analysis.
In one example embodiment, operation 126 computes the standard score (y) using
the
following formula.
y = A log(PSA) + B log(PSP94) + C.
A, B, and C, are constants. In one example, A is 2.2724, 13 is -1.4732, and C
is 0.3839. Constants
can be rounded to fewer significant digits, or expanded to greater significant
digits, in some
embodiments. Other embodiments-use other formulas or other constants, such as
those computed
from different sets of data associated with different subjects.
In general, for an equivalent number of patients categorized (i_e., for a data
set of the same
size), one would expect a database divided into three classes (healthy, having
non-malignant
disease of the prostate, having prostate cancer) to have a greater diagnostic
accuracy when used for
diagnosing patients, as compared to a database divided into six classes
(healthy, having non-
malignant disease of the prostate, having localized cancer of the prostate,
having metastasised
cancer of the prostate, having precancerous prostatic lesions, and having
acute or chronic
inflammation of prostatic tissue)- One would also reasonably expect that an
increase in the data
27

CA 02747184 2011-07-22
characterized number of patients entered into the database) would result in an
improvement in
the diagnostic accuracy of the database. Some embodiments are used for the
differential diagnosis
of any two or more of the six classes described herein.
Figure 3 is a schematic block diagram illustrating an architecture of an
example computing
device 200 for implementing various aspects according to the present
disclosure. The computing
device 200 can be used to perform some or all of one or more of the methods,
operations,
computations, or processes discussed herein, such as those illustrated and
described herein with
reference to Figures 1-2. In addition, some embodiments include two or more
computing devices
that operate together to perform aspects disclosed herein.
In one example, computing device 200 is a personal computer. Other embodiments
include other computing devices 200, such as a tablet computer, a smart phone,
a personal digital
assistant (PDA), or other device configured to process data instructions. In
some embodiments,
computing device 200 is an example of programmable electronics. In another
possible
embodiment, two or more computing devices 200 collectively form at least a
portion of the
programmable electronics:
Computing device 200 includes, in some embodiments, at least one processing
device 202
and memory 204. A variety of processing devices 202 are available from a
variety of
manufacturers, for example, Intel or Advanced Micro Devices. In some
embodiments, the
processing device 202 is configured to perform one or more methods or
operations as defined by
instructions stored in a memory device. Examples of such methods and
operations are described
herein.
Computing device 104 also includes, in some embodiments, at least one memory
device
204. Examples of memory devices 204 include read-only memory 208 and random
access
memory 210. Basic input/output system 212, containing the basic routines that
act to transfer
information within computing device 200, such as during start up, is typically
stored in read-only
memory 208- Memory device 204 can be a part of processing device 202 or can be
separate from
processing device 202.
in this example, computing device 200 also includes system bus 206 that
couples various
system components including memory 204 to processing device 202. System bus
206 is one of
any number of types of bus structures including a memory bus, or memory
controller, a peripheral
bus; and a local bus using any of a variety of bus architectures.
In some embodiments, computing device 200 also includes secondary storage
device 214
for storing digital data. An example of a secondary storage device is a hard
disk drive- Secondary
storage device 214 is connected to system bus 206 by secondary storage
interface 216. Secondary
storage devices 214 and their associated computer readable media provide
nonvolatile storage of
computer readable instructions (including application programs and program
modules), data
structures, and other data for computing device 200-
28

CA 02747184 2011-07-22
Although the exemplary architecture described herein employs a hard disk drive
as a
secondary storage device, other types of computer readable media are included
in other
embodiments. Examples of these other types of computer readable media include
magnetic
cassettes, flash memory cards, digital video disks, Bernoulli cartridges,
compact disc read only
memories, digital versatile disk read only memories, random access memories,
read only
memories, or other memory devices.
A number of program modules can be stored in secondary storage device 214 or
memory
204, including operating system 218, one or more application programs 220,
other program
modules 222, and program data 224. In some embodiments, program modules
include data
instructions that are stored in computer readable media (such as computer
readable storage media).
The data instructions, when executed by the processing device 202, cause the
processing device
202 to perform one or more of the methods or operations described herein-
In some embodiments, a user provides inputs to the computing device 200
through one or
more input device.; 230. Examples of input devices 230 include keyboard 232,
mouse 234,
touchpad 236 (or a touch sensitive display), and microphone 238. Other
embodiments include
other input devices 230. Input devices 230 are often connected to the
processing device 202
through input/output interface 240 that is coupled to system bus 206. These
input devices 230 can
be connected by any number of input/output interfaces, such as a parallel
port, serial port, game
port, or a universal serial bus. Wireless communication between input devices
and interface 240 is
possible as well, and includes infrared, BLUETOOTHHH wireless technology,
802.1 lalb/g/n/z
wireless communication, cellular communication, or other radio frequency
communication
systems in some possible embodiments.
In some embodiments, a display device 242, such as a monitor, liquid crystal
display
device, projector, or touch screen display device, is also connected to system
bus 206 via an
interface, such as display adapter 244. In'addition to display device 242, the
computing device
200 can include various other peripheral devices (not shown), such as speakers
or a printer.
When used in a local area networking environment or a wide area networking
environment (such
as the Internet), computing device 200 is ;typically connected to network 252
through a network
interface or adapter 250. Other possible embodiments use other communication
devices. For
example, some embodiments of computing device 200 include a modem for
communicating across
network 252.
Computing device 200 typically includes at least some form of computer-
readable media.
Computer readable media include any available media that can be accessed by
computing device
200. By way of example, computer-readable media include computer readable
storage media and
communication media.
The term computer readable media as used herein includes computer storage
media.
Computer storage media includes volatile and nonvolatile, removable and non-
removable media
29

CA 02747184 2011-07-22
implemented in any method or technology for storage of information, such as
computer readable
instructions, data structures, program modules, or other data. Computer
readable storage media
includes, but is not limited to, read-only memory 208, random access memory
210, electrically
erasable programmable read only memory, flash memory or other memory
technology, compact
disc read only memory, digital versatile disks or other optical storage,
magnetic cassettes,
magnetic tape, magnetic disk storage or other magnetic storage devices, or any
other medium that
can be used to store the desired information and that can be accessed by
computing device 200. In
some embodiments, computer readable storage media is non-transitory media.
Communication media can be embodied by computer readable instructions, data
structures, program modules or other data in a modulated data signal, such as
a carrier wave or
other transport mechanism and includes any information delivery media. The
term "modulated
data signal" refers to a signal that has one or more of its characteristics
set or changed in such a
manner as to encode information in the signal. By way of example,
communication media
includes wired media such as a wired network or direct-wired connection, and
wireless media such
as acoustic, radio frequency, infrared, and other wireless media- in some
embodiments,
communication media is transitory media. Combinations of any of the above are
also included
within the scope of computer readable media.
A database 260 is also illustrated in Figure 3. In some embodiments, database
is a
separate device from computing device 200, and is in data communication with
computing device
200, such as across network 252. In another possible embodiment, however,
database 260 is a
memory device that is part of computing device 200. In some embodiments,
database 260
includes a medical records database. In another embodiment, database 260
includes subject data,
such as the compilation of data for a plurality of subjects. Such data can
include, for example, one
or more of the following for each subject: PSA values, PSP94 values, age,
blood pressure values,
and Gleason Score values.
The present invention is further illustrated by the following examples, which
should not be
construed as limiting in any way.
EXAMPLES
Exam le X. Samples Used for BiomarkerDiseovery
Patients were recruited through a series of urological clinics and hospitals
located in
southern British Columbia (2 sites), Quebec (1 site), Manitoba (I site), Nova
Scotia (I site) and
Ontario (15 sites) for a pre-biopsy screening evaluation, Spot urine samples
were collected
without a preceding DRE. 24-hour urine samples were obtained prospectively no
more than ten
days prior to the patient undergoing a previously scheduled biopsy of the
prostate for suspicion of

CA 02747184 2011-07-22
prostate cancer. Serum samples were obtained by standard blood draw and
collected as a 10 mL
sample volume.
Patients were recruited for sample collection for the Pre-Biopsy Screening
provided they
were able to meet the following criteria:
= Patient was male, at least 50 years of age and able to understand, and is
willing to sign
a written informed consent document.
= Patient was previously scheduled for a biopsy of the prostate for suspicion
of prostate
cancer.
= Patient could provide urine samples for analysis and serum samples for total
PSA
testing.
= Patient had complete medical history information available (including tumour
stage
and grade if the patient was subsequently diagnosed as having prostate
cancer).
Patients were excluded when,
= Patient reported a previous incidence of prostate cancer.
= Patient reported a previous incidence of non-prostate cancer except basal
skin cell
carcinoma in the previous two years.
= Patient reported taking either investigational agents or any prescribed pre-
operative
medications at the time of sample collection.
Patient ClinicalNledical History information
Medical history information was obtained as close to the time of sample
collection as
possible. This information included: age of patient; circulating PSA levels at
time of sample
collection; pathology and history of prostate cancer; presence of other
chronic or acute conditions
unrelated to prostate cancer at the time of sample collection and current
management as well as
current and past treatment regimes for prostate cancer.
Sample Groups
Aggressive prostate cancer was defined as Gleason score of r 7 and non-cancer/
non-
aggressive cancer was defined as Gleason score 6. Non-cancer samples included:
a0 Patients diagnosed with a non-malignant disease of the prostate (for
example, benign
prostatic hypetplasia): confirmation of the absence of prostate cancer was
evaluated by
histological examination of prostatic tissue (needle point biopsy).
= Prostatic intraepithelial neoplasia (PIN) samples: patients were diagnosed
as having the
disease by confirmation of the presence of PIN through post-surgical
histological
evaluation (biopsy).
31

CA 02747184 2011-07-22
= Non-PCa/PIN samples: patients were diagnosed as being free of disease by
confirmation
of the absence of prostate cancer/PiN as evaluated by histological examination
of
prostatic tissue (biopsy)-
* Control samples- patients with no reported complaints or symptoms related to
prostate
cancer, and who were not suffering from severe disease at the time of
collection.
Table 1. Patient Samples test for PSP94 Total PSA values from 2.5 to
ng/rT.
Non-
Cancer/Non-Aggressive Non Total
Aggressive Cancer Cancer
Prostate Gleason .
Cancer score 6
Total
Samples in
Study 18 26 44 88
Men without
hypertension 7 15 22 44
Sample Handling
10 Samples originating from sites outside of Winnipeg, Manitoba were shipped
frozen on dry
ice. Those samples obtained from Victoria General Hospital.(Winnipeg,
Manitoba) were frozen at
the site and then transported on dry ice to the laboratory. Those samples
obtained from the
Winnipeg Clinic were stored at 4 C at the site for same-day pickup- These
samples were then
transported on ice to the laboratory. Upon receipt urifle samples stored at -
20 C.
Example 2; Immunoassay of Urine PSP94 in a Microspherc Multiplex System
Of 4 peaks detected by mass spectrometry in the biomarker discovery set of
samples, only
1 was statistically significant both in the original set or samples and the
validation set for both
Control vs PCa and BPH vs PCa. Prostate specific protein 94 (PSP94) with a
predicted mass of
10,770 Da was identified by LC-MSMS as the one biomarker of 10,750 Da in the
validation
samples and it co-eluted during purification with the biomarker of-.10,657 Da.
After identifying PSP94 as the biomarker of interest form mass spectrometry,
we created a
sandwich immunoassay to detect PSP94 on a l3ioPlexQD platform anti
characterized the analytic
performance of the test.
32

CA 02747184 2011-07-22
Microsphcre Prearation:
Mierospheres were coated with 10.tg polyclonal anti-PSP94 antibody (R&D
Systems,
Minneapolis, MN) per 1,250,000 beads using the BioRada coupling procedure for
the Amine
Coupling Kit.
Preparation of Calibrators and Controls:
PSP94 obtained from R&D Systems was diluted in Assay Buffer to create a 1000
ng/mL
stock solution. Dilutions were made from the 1000 ng/mL stock to create
calibrators and controls
that ranged from 0.5 ng/mL to 12 ng/mL.
Sample Prngparation before TestinQ_
Aliquoted urine samples were stored at -20 C. On the day of immunoassay
testing, the
thawed samples were centrifuged for 4 minutes at 13,000 RPM (16,000 x g) on a
lieraeus
Biofiige. The samples were diluted to 1:20 in a PBS Buffer containing bovine
serum albumin
(assay buffer).
Immugoassav to Detect of PSP94 in TAine:
This assay used a quantitative sandwich enzyme immunoassay format.
1. The polyclonal antibody specific for PSP94 was coupled onto microspheres,
and the
microspheres were vortexed and sonicated. Fifty microliters (50 L) of
resuspended
microspheres were then pipetted into a Milliporea microfilter plate and washed
with PBS
and 0.05% Tween' buffer ('Wash Buffer).
2. The washed beads were followed by the addition of 50 1iL of either PSP94
calibrators (0 to
12 ng/mL), controls (2 or 6 ng/ml) or urine samples diluted into Assay Buffer.
PSP94
present in urine bound to the polyclonal antibody attached to the
microspheres.
3. After washing away unbound substances with Wash Buffer, a 1:400 dilution of
a mouse
anti-PSP94 monoclonal antibody (Novus Biologicals) in Assay Buffer was added
to each
well-
4. After washing away unbound substances with Wash Buffer, a 1:100 dilution of
a goat anti-
mouse antibody linked to phycoerythrin (Jackson In]munoresearch) in Assay
Buffer was
added to each well. The wells were washed with Wash Buffer and resuspended in
130 uL
of Assay Buffer.
5. The filter plates were placed in the BioP1exQ 200 to quantify the
fluorescence from the
goat anti-mouse PE bound to beads. The fluorescence intensity was proportional
to the
concentration of PSP94 in urine.
6. Quantitation of results were determine by a 4-PLC curve fit from
33

CA 02747184 2011-07-22
Example 3: Analytical Performance
Typical Calibration Curve:
The average of duplicate readings for each calibrator control and sample were
calculated
using the SioPlex09 200 software to generate a 4-PLC curve fit. The zero
calibrator did not need to
be subtracted from the other calibrators, controls or samples for accurate
results. A typical curve is
shown in figure 4 and typical results for each calibrator are shown in 'Table
2.
Table 2. Typical Calibration Curve
Concentration Concentration
ng,/mL) Fluorescence %CV n ml) (Obs/Exp) 100
12 2875.3 1.17 11.22 94
8 2535 4.18 8.6 107
4 1432.5 3.55 3.99 100
1 274.3 3.74 0.97 97
0-5 125.5 4.51 0.52 103
0 25.8 4.12
Intra-assn Precision:
Controls were run in duplicate or triplicate over 13 different plates for over
I month. The
intra-assay precision for the 2 and 6 ng/mL PSP94 was less than 5% and the
inter-assay precision
is Tess than 10%.
Spike Rcovery:
An example of recovery is provided in Figure 5 where a sample with 1.6 ng/mL
PSP94 in
1;10 diluted urine was spiked with 3 and 10 ng/mL PSP94. The % recovery was
95% for spiked
samples. The slope is approximately 1.0 with a correlation coefficient of "-
1.0 indicating near
perfect recovery of PSP94 in this study.
Analytical Sensitivity:
Limit of Quantitation (LOQ) was estimated with 5 different standard assays
using multiple
bead preparations and reagent preparations. Specifically, the LOQ was
calculated by determining
the concentration of PSP94 from a 4-PLC curve fit for the
Fluorescence = mean of the zero calibrator+ 10*standard deviation of the zero
calibrator.
The I.,OQ ranged from 0.10 to 0-15 ng/mL At a 1:20 dilution, the LOQ was 2.0
to 3.0
Specifier :
PSA was added at 100 ng/mL to the zero calibrator. The concentration of PSP94
was less
than the LOQ. Therefore, PSA did not interfere with this assay.
34

CA 02747184 2011-07-22
Freeze-thaw cycles:
There was no effect (total CV from 4 - 11 % CV) of up to 4 freeze thaw cycles
on the
performance of PSP94 when tested on 5 fresh samples with PSP94 data ranging
from 8 to 220
ng/mL.
Example 4: P P94 alone and PSP94 pla PSA for the diagnosis of Prostate Cancer
Samples were collected from men according to Example 1 _ Spot urine samples
were
tested for PSP94. Matched serum samples were then analyzed for PSA. Data were
then analyzed
using MedCalc Software version 9.5.2.0 (2008) (MedCalc Software bvba,
Belgium). PSP94 on
the Bioplexil was diagnostic for Controls vs PCa; (p=0001) and BPH vs I'Ca
(p=0010) using a
pre-DRI3 spot collection. These data confirm that PSP94 was diagnostic for
prostate cancer.
We further explored the data by examining Gleason scores combined with PSP94
values
obtained from pre-DRE spot collections and 24 hr collections from the same
men. We observed
that a Gleason Score of 7, 8 and 9 had decreased values of PSP94 compared to a
Gleason Score of
6 or noncancer_ An example of this is shown in Figure 5 for PSP94 at 24 hr.
When we examined
points that were above the whiskers in Figure 5, we observed that 5 of 6
patients had hypertension.
No other disease correlated as well for these samples.
Table 3: Six patients that had elevated PSP94 above the whiskers for Gleason
score 7 and 8.
Patient Medical History Mods Gleason Score
No
Detrol LA, Norvasc, Avandia,
2016 Hypertension, high cholesterol, Glyburide, Novo-Hydrazide. 4+4=8
diabetes, BPH, glasses Metrormin, Vasotec, Novo-
Terazosin. Lipitor. Betoptic w
Hypertension; gastrointestinal
(Diverticulitis); has trouble with Parfet (gastric reflex; 20mg 00);
organism, Asthma, Decreased flow of HCT2 (hypertension 12.5mg);
2015 urine, Allergies to flagyl_ trees, grasses, Synthroid (thyroid 0.05mg);
4'r3=7; 4110
Depression. Benign basil skin Symbicant (asthama); Calcium positive cores
carcinoma (7 yrs ago), grandmother and puffer (general bone health);
mother had breast cancer, father had Cipro (gastrointestinal flare-ups)
prostate cancer. sister had bone cancer
pulmonary embolism-1996, phlebitis- Cournadin, Nexium, Exetrol,
2092 1996. hypercholesteralemia, heart burn, 3+4=7
Sister skin cancer on scalp 2ocor, AcetanlinOphene
Hypertension, heart burn. protatis,
vasectomy. elevated cholesterol, frozen
2099 shoulder, itchy head, allergies. S Aelexin,nLipitor, Norvasc, Periet,
3+4=7
hemmoroids, inflammed groin, fell and ele, viLaml1 B, Vitamin C
had a bruised testicle

CA 02747184 2011-07-22
Hypertension, high cholesterol,
indigestion, Hernia, high iron (in blood). Arthrofec, ASA. Betamethasone,
2160 psorasis, , Mother breast cancer, Father Coversyl. F-locom cream,
Lipitor, 3+4=7
liver cancer, Brother brain & bowel Losec, Metformin, Tylenol 3
cancer
2163 Hypertension; allaries to MSG Pariet, Hydroxyquine, Naproxen, 4+3=7&
4+4=8
Vitamin 0, Prednisone
Based on these observations, we wanted to determine if hypertension interferes
with
PSP94 quantitation. To test whether hypertension interferes, we used the area
under the curve
(AUC) for the ROC curve to examine the diagnostic capability of the test. An
AUC of 1.0 is
diagnostically perfect, and an AUC of 0.50 is has no diagnostic capability-
Therefore, an AUC
that is statistically significant above 0.50 indicates an improvement in
diagnostic capability of the
test with values close to 1 A having a higher diagnostic significance. When we
examined the
ability of PSP94 in spot collected urine to diagnose prostate cancer or high
Gleason Score (HGS)
from non-cancer patients, we observed that men without hypertension had
statistically significant
AUC above 0.50, which indicated the diagnostic capability of the test. In
contrast, men with
hypertension did not have statistically significant AUC for note-cancer
compared to prostate cancer
(0.612) or non-cancer versus HGS (0.626). PSP94 samples from urine collection
at 24 hr had
similar results as the PSP94 Spot Collection. PSP94 in urine samples at 24 hr
from non-
hypertensive males were diagnostic for non-cancer versus prostate cancer
(0.680) and non-cancer
5 versus I-IGS (0.847) but were not diagnostic for men with hypertension-
Table 4. The area under the curve (AUC) for PSP94 and PSA when examining the
diagnostic
effectiveness of non-cancer compared to either prostate cancer or high Gleason
score when men
were categorized as having either normal blood pressure or hypertension.
PSP94 Spot Collection PSP94 24 hr PSA
n L) Collection (n /mL) (n?!mL)
Normal Normal Normal
Blood Hyper- Blood Hyper- Blood Hyper-
Pressure tension Pressure tension Pressure tension
noncancer vs
PCa 0.711' 0.612 0.680 0.572 0.664' 0.739`
Noricancer vs
HGS 0.773' 0.626 0.847` 0.596 0.726 0.757-
* p,0.05
36

CA 02747184 2011-07-22
A possible explanation for the distinction between men with and without
hypertension was
sampling bias. To eliminate the possibility of sampling bias, we demonstrated
that another
biomarker in the same men did not demonstrate the observed "hypertension
effect-" Therefore, we
used a general screening test to measure total PSA measurements as a control
to verify that the
data set was not biased. Table 4 indicates that PSA is statistically
diagnostic in non-cancer versus
prostate cancer and non-cancer versus HGS in men with or without hypertension.
Thus, the lack
of diagnosis for prostate cancer by measuring PSP94 is not due to a bias in
the data and strongly
indicates that hypertension affects PSP94 measurements.
Next we next explored the ability of PSP94, PSA, age, smoking history and BMl
to be
combined for potential screening tests. There was also no significant
correlation between PSP94,
PSA, age, smoking history and BMI with the exception of a weak (r-- -0.205)
correlation between
BMI and age. Thus, it was possible to combine these diagnostic and clinical
characteristics with
logistic regression to improve diagnostic capabilities. For logistic
regression, we used log(PSA)
and log(PSP94) to normalize the data first before analysis. We examined HGS
and PCa diagnoses
with the algorithms created by logistic regression. Improvements in the AUC
for the ROC curves
were observed (Table 5) when compared to either PSA or PSP94 results alone.
Table 5. AUC for the ROC curves from logistic regression models for the
diagnosis of non-cancer
from either PCa or PIGS using PSA and PSP94 in men with normal blood pressure.
PSP94 Spot and PSA PSP94 24 hr
Collection (n 1ML) Collection n /mL
Normal Blood Normal Blood
Pressure Pressure
noncancer vs PCa 0.778 0.714
Noncancer vs High
GleasonScore* 0.881 0.881
All models AUC had p<0.0001 compared to AUC=0.50.
For example, the AUC of the ROC curve for the logistic regression of PSP94
spot
collection and PSA for i-IGS versus noneancer samples from men with normal
blood pressure
were statistically higher compared to either PSP94 spot collection (p= 0.048)
or PSA (p=0.013)
alone (Figure 6). These data indicate that PSP94 and PSA results can be
combined to improve the
diagnosis 1-IGS compared PSA alone. This improvement is demonstrated in Table
6 where a-2.5
fold improvement in prostate cancer detection with logistic regression model
of PSP94 and PSA
compared to PSA alone.
37

CA 02747184 2011-07-22
Table 6. Detection in Prostate Cancer at Specificity of -77% by PSA or
algorithm of PSP94 spot
collection and PSA.
Gleason
Non Cancer Score 6 HGS
Cutoff TN/ TN+FN TP/(TP+FP TP/ TP+FP
PSA 7 >10 ng/ml 35/45 (78%) 1 4/22(18%) 9120 45%
Algorithm ? 1-2848 34145 (76%) 12122(55% 18/20 90%
TP = true positive; FP = false positive; IN = true negative; ~N:= Use negative
From Table 6, we were able to provide two flow charts of expected results from
100 men
going to a urology clinic in Canada. Figure 7 demonstrates the current PSA
test with a high level-
of specificity which exists at= 10 ng/mL that would decrease the number of
unnecessary biopsies.
Under these conditions, PSA would miss 33 of 48 men with cancer- PSA nonnally
is selected at 4
ng/mL to detect men with cancer, however, it cones at the expense of a >70% of
unnecessary
biopsies. However, when we combined PSA with PSP94 spot collection, we
achieved both high
sensitivity and specificity where we detected 90% of all cancers in high
Gleason score and the
majority of Gleason score 6 samples. The diagnostic capability of the
algorithm is the total correct
diagnoses compared to the total samples tested. The diagnostic capability of
the algorithm is 74%
compared to PSA at 55%.
We also examined the diagnosis of PCa and HGS with PSP94 in men with <i0 ng/mL
PSA_ This cutoff was selected since men with > 10 ng/mL PSA are normally
selected for biopsy-
The hypothesis is that PSP94 would improve the diagnosis of high Gleason score
prostate cancer
compared to men with 0 to 10 ng/mL PSA and the results are shown in Table 7.
Men with 510
ng/mL PSA would benefit from improvements with a PSP94 test where PSP94 in
spot collection
(p=0.017) and PSP94 in 24 hr urine (p=0.009) had statistically significant
improvement in the
AUC compared to PSA. PSP94 spot collection and PSP94 in 24 hr urine where the
PSP94 in 24 hr
urine is close to an AUC of 1-0 and has a 100% sensitivity and 78%
specificity. When 100 men
with normal blood pressure and with PSA <10 ng/mL go to a Canadian clinic, 54
men would have
unnecessary biopsies if being tested for high Gleason Score prostate cancer
and 4 men would not
be detected for HGS with a PSA cutoff value of>4.0 ng/mL. This is in contrast
to PSP94 in 24 hr
urine where only 15 unnecessary biopsies and detected all FIGS when PSI'94 in
24 fir urine was
<28.7 ng/mL. When we examined the capability of separating HGS prostate cancer
from all other
samples in men with PSA values below 10.00 ng/mL and normal blood pressure, we
observed a
85% diagnostic capability for PSP94 in 24 hr urine collection while PSA from 0
to 10.0 ng/mL had
a 42% diagnostic capability and the number of unnecessary biopsies would
decreases from 54 with
PSA to 15 with PSP94.
38

CA 02747184 2011-07-22
Table 7_ AUC for the ROC curves from data with PSA values from 0-10.0 ng/ml,
from men with
normal blood pressure.
PSP94 Spot 24 hr Collection PSA
Collection (ng/rL) (nh/mL) (rig/mL)
noncancer vs PCa 0.694 0.680 0.708
Noneancer vs High
Gleason Score 0.833 0.914 0.723
In these studies, we observed that both PSP94 values from spot collection and
PSP94 24
hour collection can be combined with results from PSA to improve screening for
men with high
Gleason score (?7) compared to non-cancer. This can occur by creation of
algorithms of PSA with
PSP94 in spot collection or by the selecting patients with PSA values from 0
to 10 ng/mL. The
combination of PSP94 with PSA can identify high Gleason score patients most at
risk for
aggressive prostate cancer and also reduce the number of unnecessary biopsies
in men. PSP94 test
in combination with PSA would also be useful in active surveillance where men
have low Gleason
score prostate cancer but would like an non invasive test to determine when
they are at risk for
more aggressive prostate cancer at higher Gleason scores (>= 7) and thus
minimize the number of
biopsies required in the future_
39

Representative Drawing