U.S. patent application number 10/414989 was filed with the patent office on 2003-11-20 for non-invasive enzyme screen for cancer.
Invention is credited to Freeman, Michael R., Moses, Marsha A., Wiederschain, Dmitri.
Application Number | 20030215900 10/414989 |
Document ID | / |
Family ID | 33303366 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030215900 |
Kind Code |
A1 |
Moses, Marsha A. ; et
al. |
November 20, 2003 |
Non-invasive enzyme screen for cancer
Abstract
Methods and kits for diagnosing the presence of and prognosing
the appearance of tissue remodelling-associated conditions,
involving the presence of enzymes in a biological sample, are
disclosed. In particular, the method pertains to diagnosing the
presence of or prognosing appearance of cancer, metastatic cancer,
and obstructive and degenerative conditions.
Inventors: |
Moses, Marsha A.;
(Brookline, MA) ; Freeman, Michael R.; (Boston,
MA) ; Wiederschain, Dmitri; (Brookline, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
33303366 |
Appl. No.: |
10/414989 |
Filed: |
April 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10414989 |
Apr 16, 2003 |
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09469637 |
Dec 22, 1999 |
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09469637 |
Dec 22, 1999 |
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08843095 |
Apr 25, 1997 |
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6037138 |
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08843095 |
Apr 25, 1997 |
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08639373 |
Apr 26, 1996 |
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Current U.S.
Class: |
435/23 ;
435/7.23 |
Current CPC
Class: |
G01N 33/573 20130101;
G01N 33/57434 20130101; G01N 33/57415 20130101; G01N 2333/96486
20130101; G01N 33/574 20130101; G01N 33/564 20130101; G01N 33/57488
20130101 |
Class at
Publication: |
435/23 ;
435/7.23 |
International
Class: |
G01N 033/574; C12Q
001/37 |
Claims
1. A non-invasive method for facilitating the diagnosis of a
subject for a tissue remodelling-associated condition, comprising:
obtaining a urine sample from a subject; and detecting an enzyme in
the urine sample, thereby facilitating the diagnosis of the subject
for the tissue remodelling-associated condition.
2. The method of claim 1, wherein the tissue remodelling-associated
condition is cancer.
3. The method of claim 1, wherein the tissue remodelling-associated
condition is an arthritic condition, an obstructive condition, or a
degenerative condition.
4. The method of claim 2, wherein the cancer is organ-confined
prostate cancer.
5. The method of claim 2, wherein the cancer is metastatic prostate
cancer.
6. The method of claim 2, wherein the cancer is in cells of
epithelial origin.
7. The method of claim 6, wherein the cancer is selected from the
group consisting of cancers of the nervous system, breast, retina,
lung, skin, kidney, liver, pancreas, genito-urinary tract, and
gastrointestinal tract.
8. The method of claim 2, wherein the cancer appears in cells of
mesodermal origin.
9. The method of claim 2, wherein the cancer appears in cells of
endodermal origin.
10. The method of claim 2, wherein the cancer affects cells of bone
or of hematopoietic origin.
11. The method of claim 1, wherein the enzyme is involved in a
pathway of tissue remodelling or reshaping.
12. The method of claim 1, wherein the enzyme is a matrix-digesting
enzyme.
13. The method of claim 1, wherein the enzyme is a protease.
14. The method of claim 13, wherein the protease is a serine
protease.
15. The method of claim 13, wherein the protease is a matrix
metalloproteinase.
16. The method of claim 1, wherein the enzyme is a proenzyme.
17. The method of claim 1, further comprising removal of low
molecular weight contaminants from the urine prior to the detection
step.
18. The method of claim 17, wherein the urine is dialyzed.
19. A non-invasive method for facilitating the diagnosis of a
subject for a disorder of the prostate, comprising: obtaining a
urine sample from a subject; and detecting a prostate
disorder-associated enzyme in the urine sample, thereby
facilitating the diagnosis of the subject for the prostate
disorder.
20. The method of claim 19, wherein the prostate-disorder
associated enzyme is a matrix-digesting enzyme.
21. The method of claim 19, wherein the matrix-digesting enzyme is
a protease.
22. The method of claim 21, wherein the enzyme is a
metalloproteinase.
23. The method of claim 19, wherein the disorder of the prostate is
benign prostatic hyperplasia.
24. The method of claim 19, wherein the disorder of the prostate is
organ-confined prostate cancer.
25. The method of claim 19, wherein the subject has previously been
treated surgically or hormonally.
26. The method of claim 25, wherein the subject has been treated to
block testosterone.
27. The method of claim 19, wherein the disorder is metastatic
cancer.
28. A method for facilitating the diagnosis of a subject for
prostate cancer, comprising: obtaining a urine sample from a
subject suspected of having prostate cancer; and detecting a
prostate cancer-associated enzyme in the urine sample, thereby
facilitating the diagnosis of the subject for prostate cancer.
29. The method of claim 28, wherein the prostate cancer-associated
enzyme is a protease.
30. The method of claim 29, wherein the protease is a matrix
metalloproteinase.
31. The method of claim 30, wherein the matrix metalloproteinase is
gelatinase A or gelatinase B.
32. The method of claim 28, wherein the subject has benign
prostatic hyperplasia.
33. The method of claim 28, wherein the subject is under treatment
to block testosterone.
34. The method of claim 28, further comprising removal of low
molecular weight contaminants from the urine prior to the detection
step.
35. A method for facilitating the prognosis of prostate cancer in a
subject, comprising: obtaining a biological sample from a subject;
and detecting a prostate cancer-associated enzyme, thereby
facilitating the prognosis of prostate cancer in a subject.
36. The method of claim 35, wherein the biological sample is
urine.
37. The method of claim 35, wherein the prostate cancer
associated-enzyme is a tissue remodelling-associated enzyme.
38. The method of claim 37, wherein the prostate-cancer associated
enzyme is a protease.
39. The method of claim 38, wherein the protease is a type IV
collagenase.
40. The method of claim 39, wherein the metalloproteinase has a
molecular weight of approximately equal to or greater than 82 kDa
or 92 kDa.
41. The method of claim 39, wherein the metalloproteinase has a
molecular weight of approximately 72 kDa.
42. The method of claim 35, wherein the subject has benign
prostatic hyperplasia.
43. A method for prognosis of problematic prostatic hyperplasia in
a subject, comprising: obtaining a biological sample from a
subject; and detecting a problematic prostatic
hyperplasia-associated enzyme in the biological sample, thereby
facilitating the prognosis of problematic prostatic hyperplasia in
a subject.
44. The method of claim 43, wherein the prostatic
hyperplasia-associated enzyme is a metalloproteinase.
45. The method of claim 44, wherein the metalloproteinase has a
molecular weight of approximately equal to or greater than 92
kDa.
46. A method for prognosis of metastatic prostate cancer
comprising: obtaining a biological sample from a subject; and
detecting a metastatic prostate cancer-associated enzyme in the
biological sample, thereby facilitating the prognosis of metastatic
prostate cancer in a subject.
47. The method of claim 1, wherein the enzyme has a molecular
weight of approximately 72 kDa or approximately 92 kDa.
48. The method of claim 1, wherein the enzyme has a molecular
weight equal to or greater than approximately 150 kDa.
49. The method of claim 43 or 46, further comprising removal of low
molecular weight contaminants from the urine prior to the detection
step.
50. The method of claim 1, wherein the enzyme is detected
electrophoretically.
51. The method of claim 50, wherein the electrophoretic pattern is
a zymogram.
52. The method of claim 51, wherein the zymogram substrate is
gelatin, casein, fibronectin, vitronectin, plasmin, plasminogen,
type IV collagen, or a derivative of type IV collagen.
53. The method of claim 1, wherein the enzyme is detected
immunochemically.
54. The method of claim 53, wherein the enzyme is detected by a
radio-immune assay.
55. The method of claim 53, wherein the enzyme is detected by an
enzyme-linked immunosorbant assay.
56. A kit for facilitating the diagnosis and prognosis of a tissue
remodelling-associated condition, comprising: a container having a
reagent for detecting an enzyme in a urine sample; and instructions
for using said reagent for detecting the enzyme for facilitating
the diagnosis and prognosis of a tissue remodelling-associated
condition.
57. The kit of claim 56, wherein the tissue remodelling-associated
condition is cancer.
58. The kit of claim 56, wherein the tissue remodelling-associated
condition is an arthritic condition, an obstructive condition, or a
degenerative condition.
59. The kit of claim 57, wherein the cancer is organ-confined
prostate cancer.
60. The kit of claim 57, wherein the cancer is metastatic prostate
cancer.
61. The kit of claim 56, wherein the enzyme is a matrix
metalloproteinase.
62. The kit of claim 61, wherein the matrix metalloproteinase is a
gelatinase.
63. The kit of claim 56, further comprising an apparatus for
separating urine into components for removal of low molecular
weight contaminants.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
Ser. No. 08/639,373 filed on Apr. 26, 1996, now pending. The
contents of the aforementioned application is expressly incorprated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] A class of disorders may be characterized as tissue
remodelling-associated conditions, and includes cancers, arthritic
conditions, obstructive disorders, degenerative disorders, and
problematic wound-healing and ulcerative disorders. Paradigmatic
among these is prostate cancer (CaP), the leading source of new
cases of cancer in men in this country, and the second leading
cancer cause of cancer death after lung cancer. Over 40,000
Americans are estimated to have died of CaP in 1995, and about
244,000 new cases of prostate cancer were detected (Cancer Facts
and Figures--1995, American Cancer Society, Inc., 1995) and these
numbers have increased annually at an alarming rate. Further, the
rate of appearance of prostate cancer in African-American men is
37% higher than for their white counterparts (Jaroff, L. (Apr. 1,
1996), Time).
[0003] The current primary diagnostic tool for disorders of the
prostate is measurement of the level of prostate-specific antigen
(PSA) in blood, which in normal men ranges from 0 to 4
nanograms/milliliters. Prostate enlargement, a condition known as
benign prostatic hyperplasia (BPH), is found in about half of men
over age 45. With BPH, PSA levels rise in proportion to prostate
size, possibly obscuring diagnosis of CaP. In addition, a
significant proportion of men with CaP have normal PSA levels. The
PSA test is somewhat non-specific for distinguishing CaP and BPH,
and produces a degree of false negative results (Garnick, M.,
(1993), Am. Inst. Med, 118:804-818). Further, the PSA test is
somewhat invasive, requiring the subject to give a blood sample, a
procedure that requires trained personnel, in the setting of a
doctor's office or clinic. The PSA test, a major advance over
previous procedures, thus leaves much to be desired.
[0004] CaP is treated by surgery, radiation therapy, cryotherapy or
implantation of radioactive seeds, or a combination of these
procedures. In choosing one of these treatments, consideration is
also taken of possible sequelae that impact negatively on quality
of life, such as temporary or long-term incontinence and impotence.
Further, following surgical or chemotherapeutic treatment,
production of testosterone is suppressed hormonally, to discourage
metastases in the CaP patient. Hormonal suppression is found to be
effective for several years duration, however cancer cells that
have metastasized eventually become resistant to the drugs of
hormonal suppression. CaP metastasis to other sites is inevitably
fatal. Only a small percent of men with microscopically-detectable
CaP progress to metastatic cancer and actually die of this disease.
A current medical approach, particularly for the elderly, is
"watchful waiting", wherein tumors are not treated but rather
monitored for progression.
SUMMARY OF THE INVENTION
[0005] The present invention provides biological markers to
non-invasively monitor the diagnosis and prognosis of prostate
disorders and other tissue remodelling-associated conditions. This
invention provides methods and kits using non-invasive procedures
for detection of tissue remodelling-associated conditions in
subjects and patients, for diagnosis of diseases such as prostate
cancer, breast cancer, ovarian cancer, brain tumors, arthritic
conditions, obstructive conditions, and ulcerative conditions. The
primary screens use biological fluid samples that may be obtained
by personnel without medical training, and do not require visiting
a clinic or hospital. The statistical association between positive
results and occurrence of tissue remodelling-associated conditions
are applied to early diagnoses of the appearance of these
conditions, and to prognoses of changes in these conditions.
[0006] The present invention features non-invasive methods for
facilitating diagnosis of a subject for a tissue
remodelling-associated condition. The method involves obtaining a
biological sample from that subject and detecting an enzyme in that
sample, facilitating the diagnosis. The non-invasive method was
based at least in part, on the observation of full length, active
intact enzymes that are normally associated with the process of
tissue remodelling, in urine samples from patients with certain
conditions. For example, gelatin-degrading matrix
metalloproteinases and other proteases have been found in the urine
of cancer patients. Further, high statistical associations between
presence of prostate cancer and appearance of certain enzymes in
urine, and between metastatic cancer and certain enzymes in urine,
have been found.
[0007] The tissue remodelling conditions that can be monitored by
the methods of this invention include a variety of types of cancer;
moreover, the enzymes are suitable for diagnosis of other tissue
remodelling conditions, such as arthritis, degenerative conditions,
and obstructive conditions. The invention provides non-invasive
methods for diagnosing these conditions by assay for enzymes in
biological fluids.
[0008] More preferably, the methods of this invention embody
detection of enzymes in urine, for diagnosis and prognosis of
cancer, and most preferably, prostate cancer. The invention also
relates to diagnosis and prognosis of metastatic prostate cancer.
The varieties of cancer suitable for diagnosis by the methods of
this invention include, among others, cancers of epithelial origin,
for example, cancers of the nervous system, breast, retina, lung,
skin, kidney, liver, pancreas, genito-urinary tract, ovarian,
uterine and vaginal cancers, and gastrointestinal tract cancers,
which form in cells of epithelial origin. Using the methods
described here, cancers of mesodermal and endodermal origin, for
example, cancers arising in bone or in hematopoietic cells, are
also diagnosed.
[0009] In a preferred embodiment, the enzymes that are detected are
matrix-digesting enzymes, more preferably, enzymes that are
proteinases, and most preferably, enzymes that are
metalloproteinases. In a different aspect, the methods of this
invention involve enzymes that are full-length active enzymes, and
they are matrix metalloproteinases. In another aspect, the method
involves removal of low molecular weight contaminants from urine
prior to the detection step; preferably, the urine is dialyzed to
remove low molecular contaminants prior to the detection step.
[0010] Another aspect of the methods features a fully non-invasive
means for facilitating the diagnosis of a subject for a disorder of
the prostate. A urine sample is obtained from the subject, and a
prostate disorder-associated enzyme is detected in the urine
sample, facilitating the diagnosis of that subject for the prostate
disorder. More preferably, the prostate disorder-associated enzyme
is a matrix-digesting enzyme, most preferably, a proteinase which
is a metalloproteinase. The disorders of the prostate include
benign prostatic hyperplasia, "problematic" prostatic hyperplasia,
organ-defined prostate cancer, this cancer which may previously
have been treated surgically or chemically, and particularly,
situations in which metastatic cancer is suspected. The method
encompasses diagnosis of subjects who are being treated hormonally
with agents that block testosterone.
[0011] The invention facilitates diagnosis of subjects for prostate
cancer, using a urine sample from such subjects, and detecting one
or more prostate cancer-associated enzymes. Enzymes of the matrix
metalloproteinase class are among those that are diagnostic, and in
the case of prostate cancer, the method involves detection of
gelatinase A, gelatinase B, and related activities. More
preferably, the detected metalloproteinase enzyme has a molecular
weight approximately equal to 72 kDa, 92 kDa, or equal to or
greater than approximately 150 kDa. Yet another feature of the
invention is a method for prognosis of metastatic prostate cancer,
by obtaining a biological sample from a subject and detecting a
metastatic prostate cancer-associated enzyme in that biological
sample facilitating the prognosis of metastatic prostate cancer. In
the preferred embodiment for detecting these enzymes, low molecular
weight contaminants are removed from the urine prior to the
detection step.
[0012] Detection of enzymes in biological fluids may be by
electrophoresis, and a preferred method of analysis of the
electrophoretogram is to develop a pattern of enzyme migration
mobilities as a zymogram. The zymogram involves incorporating an
enzymatic substrate into the inert matrix in which the enzyme
species migrate. Examples of suitable substrates are type IV
collagen or a derivative of a type IV collagen, and in the Examples
used here, the substrate is the collagen derivative gelatin. Other
convenient protein substrates, e.g., casein, are also encompassed
by the methods of the invention. Other methods of enzyme detection
may be immunochemical, for example, the enzymes may be detected by
radio-immune assay or by enzyme-linked immunosorbant assay.
[0013] The invention features kits for facilitating diagnosis and
prognosis of tissue remodelling-associated conditions, which have a
container with a reagent for detecting an enzyme in a urine sample,
and instructions. In a preferred embodiment of the kit, the tissue
remodelling-associated conditions being detected are one or more
types of cancer, for example, organ-confined prostatic cancer,
metastatic cancer, and prognosis of metastasis in a prostate cancer
patient. In a different embodiment, the tissue
remodelling-associated condition is an arthritic, obstructive, or
degenerative condition.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides non-invasive methods, between
presence of enzymes in biological fluids, and diagnosis and
prognosis of tissue remodelling-associated conditions (TRACs),
especially cancers, obstructive and degenerative conditions, and
arthritic conditions, and kits for use for such diagnosis and
prognosis. Diagnoses and prognoses for TRACs have been developed
based on observed statistical associations between these conditions
and the presence of a pattern of enzymes in biological fluids. For
convenience, certain terms employed in the specification, examples
and appended claims are collected here.
[0015] The term "subject," as used herein, refers to a living
animal or human in need of diagnosis or prognosis for, or
susceptible to, a condition, in particular an "tissue
remodelling-associated condition" as defined below. The subject is
an organism capable of responding to tissue remodelling signals
such as growth factors, under some circumstances, the subject is
susceptible to cancer and to arthritis. In preferred embodiments,
the subject is a mammal, including humans and non-human mammals
such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and
mice. In the most preferred embodiment, the subject is a human. The
term "subject" does not preclude individuals that are entirely
normal with respect to tissue remodelling-associated conditions or
normal in all respects. The subject may formerly have been treated
surgically or by chemotherapy, and may be under treatment by
hormone therapy or have been treated by hormone therapy in the
past.
[0016] The term "patient," as used herein, refers to a human
subject who has presented at a clinical setting with a particular
symptom or symptoms suggesting one or more diagnoses. A patient may
be in need of further categorization by clinical procedures
well-known to medical practitioners of the art (or may have no
further disease indications and appear to be in any or all respects
normal). A patient's diagnosis may alter during the course of
disease progression, such as development of further disease
symptoms, or remission of the disease, either spontaneously or
during the course of a therapeutic regimen or treatment. In the
invention here, a patient described in the Examples is listed with
other patients according to the most recent diagnosis of the
medical condition, and any previous diagnoses, if different, are
described in the text. Thus, the term "diagnosis" does not preclude
different earlier or later diagnoses for any particular patient or
subject. The term "prognosis" refers to assessment for a subject or
patient of a probability of developing a condition associated with
or otherwise indicated by presence of one or more enzymes in a
biological sample, preferably in urine.
[0017] The term "biological sample" includes biological samples
obtained from a subject. Examples of such samples include urine,
blood taken from a prick of the finger or other source such as
intravenous, blood fractions such as serum and plasma, feces and
fecal material and extracts, saliva, cerebrospinal fluid, amniotic
fluid, mucus, and cell and tissue material such as cheek smear, Pap
smear, fine needle aspiration, sternum puncture, and any other
biopsied material taken during standard medical and open surgical
procedures.
[0018] The term "invasiveness" as used here with respect to
metastatic cancer (Darnell, J. (1990), Molecular Cell Biology,
Third Ed, W. H. Freeman, N.Y.) is distinct from the use of the term
"invasive" to describe a medical procedure, and the distinction is
made in context. "Invasive" for a medical procedure pertains to the
extent to which a particular procedure interrupts the integrity of
the body. "Invasiveness" ranges from fully non-invasive, such as
collection of urine or saliva; to mildly invasive, for example a
Pap smear, a cheek scrape or blood test, which requires trained
personnel in a clinical setting; to more invasive, such as a
sternum marrow collection or spinal tap; to extensively invasive,
such as open surgery to detect the size and nature of tumors by
biopsy of material, taken for example during brain surgery, lung
surgery, or transurethral resection in the case of prostate
cancer.
[0019] The term "invasive" is also used with respect to proclivity
of a tumor for expanding beyond its boundaries into adjacent
tissue, or to the characteristic of the tumor with respect to
metastasis (Darnell, J. (1990), Molecular Cell Biology, Third Ed.,
W. H. Freeman, N.Y.). For example, a basal cell carcinoma of the
skin is a non-invasive or minimally invasive tumor, confined to the
site of the primary tumor and expanding in size, but not
metastasizing. In contrast, the cancer melanoma is highly invasive
of adjacent and distal tissues. The invasive property of a tumor is
often accompanied by the elaboration of proteolytic enzymes, such
as collagenases, that degrade matrix material and basement membrane
material to enable the tumor to expand beyond the confines of the
capsule, and beyond confines of the particular tissue in which that
tumor is located. Elaboration of such enzymes may be by endogenous
synthesis within the tumor cells, or may be elicited from adjacent
cells or by circulating neutrophils, in which cases the elicitation
by the tumor results from chemical messengers elaborated by the
tumor and expression of the enzymes occurs at the tumor site or
proximal to the tumor. The enzymes of the present invention are not
intended to be limited to those produced exclusively as endogenous
tumor products, but are found in biological samples in patients in
need of or subjects in need of prognosis or diagnosis of TRACs.
[0020] Cancer or neoplasia is characterized by deregulated cell
growth and division. A tumor arising in a tissue originating from
endoderm or exoderm is called a carcinoma, and one arising in
tissue originating from mesoderm is known as a sarcoma (Darnell, J.
(1990), Molecular Cell Biology, Third Ed., W. H. Freeman, NY). A
current model of the mechanism for the origin of a tumor is by
mutation in a gene known as an oncogene, or by inactivation of a
second tumor-suppressing genes (Weinberg, R. A., (September 1988),
Scientific Amer.,44-51). The oncogenes identified thus far have
arisen only in somatic cells, and thus have been incapable of
transmitting their effects to the germ line of the host animal. In
contrast, mutations in tumor-suppressing genes can be identified in
germ line cells, and are thus transmissible to an animal's progeny.
Examples of cancers include cancers of the nervous system, breast,
retina, lung, skin, kidney, liver, pancreas, genito-urinary tract,
gastrointestinal tract, cancers of bone, and cancers of
hematopoietic origin such as leukemias and lymphomas. In one
embodiment of the present invention, the cancer is not a cancer of
the bladder.
[0021] An arthritic condition such as rheumatoid arthritis is an
example of a TRAC since the disease when chronic is characterized
by disruption of collagenous structures (J. Orten et al., (1982),
Human Biochemistry, Tenth Ed., C. V. Mosby, St. Louis, Mo.). Excess
collagenase is produced by cells of the proliferating synovium.
Other TRAC conditions such as ulcerative, obstructive and
degenerative diseases are similarly characterized by alterations in
the enzymes of metabolism of structural proteins.
[0022] The term "prostate cancer" (CaP) as used herein refers to
both the appearance of a palpable tumor of the prostate, and also
to microscopically detectable neoplastic or transformed cells in
the prostate gland. In the latter case, the said
cytologically-detectable prostate cancer may be asymptomatic, in
that neither the patient nor the medical practitioner detects the
presence of the cancer cells. It is estimated that 10 million
American men carry microscopic CaP (M. Garnick, (April 1994), Sci.
Am., 78). Cancer cells are generally found in the prostates of men
who live into their seventies or eighties, however not all of these
men develop prostate cancer (CaP). Autopsies show microscopic
clusters of prostate cancer cells in one-third of men who die of
other causes (Thayer, W., (Mar. 19, 1996), quoted in Nutr. Act.
Newsletter, 23(2):12). Death rates from prostate cancer rise after
age 55, and new cases of prostate cancer, are increasing even
faster than the death rate. CaP is the second leading cause of
cancer death in men, causing over 40,000 deaths in 1995. In the
event that prostate cancer metastasizes to additional sites distal
to the prostate, the condition is described as metastatic cancer
(MC), or prostate cancer, metastasized, to distinguish this
condition from organ-confined prostate cancer. CaP fatality results
from metastatic dissemination of prostatic adenocarcinoma cells to
distant sites, usually in the axial skeleton.
[0023] The term "organ-confined", as used herein, refers to
prostate cancer that has not metastasized beyond the boundaries of
the prostate gland, i.e., has not been found by techniques familiar
to those skilled in the art to occur in any organs or tissues
beyond the prostate gland. It can not be ruled out, however, that
some number of cells have metastasized, however they are not
detected by ordinary techniques used by those with skill in the
art.
[0024] The term "metastasis" as used herein refers to the condition
of spread of cancer from the organ of origin to additional distal
sites in the patient. Preferential target organs are common to
cancer types, e.g., CaP frequently metastasizes to bone, with
concomitant symptoms such as back pain and acute urinary retention,
and high levels of mortality. Metastatic cancer (MC) as exemplified
for the purposes of this invention, is not limited to spread of CaP
to bone or any particular organ, and includes also spread of other
cancers such as kidneys (renal), breast, and gastrointestinal tract
to organs beyond these primary sites.
[0025] The phrase "benign prostatic hypertrophy," as used herein,
comprises an age-related non-cancerous enlargement of the prostate,
and affects more than 50% of men over age 45 (Garnick, supra).
Benign prostatic hypertrophy (BPH) may be asymptomatic, that is,
have no negative consequences for the individual, and is not
intended here to imply the necessary development of prostate
cancer. BPH is accompanied by an increase in production of the
protein prostate specific antigen (PSA discussed infra),
proportional to the extent of growth of the prostate gland. For
this reason, the diagnosis of CaP in a BPH patient may be difficult
to distinguish from further asymptomatic growth by sole use of the
PSA test (vide infra).
[0026] BPH may appear as or may progress to "problematic" prostatic
hyperplasia, with symptoms that include urinary urgency, frequency,
and hesitancy, and penile erectile difficulties. Since these same
symptoms are associated with CaP (M. Garnick, (1993), Annals Int.
Med., 118(10):804-818), the clinician distinguishes CaP and
problematic prostatic hyperplasia by the suddenness in onset of
symptoms, and by additional diagnostic tests (described below). BPH
and problematic prostatic hypertrophy may also progress to CaP,
however these terms are meant neither to exclude nor to imply
disease progression, as the full range of diagnostic possibilities
is found for the BPH patient population as for the normal
subject.
[0027] At present, the primary diagnostic tool for prostate
disorders is a blood test that measures prostate specific antigen
(PSA) levels. Elevated PSA is associated both with CaP and with
BPH, and PSA levels also increase with age. In cases of CaP,
removal of the prostate should produce a PSA reading of zero, and a
subsequent positive PSA reading in the blood indicates that the
cancer has metastasized, a condition that is incurable and fatal.
CaP that remains organ-confined is treated primarily by surgery and
hormone therapy to block testosterone, treatments that frequently
cause a variable period of incontinence and loss of libido, and may
only temporarily block tumor growth or metastasis.
[0028] Hormone suppression of recurrence and metastasis of prostate
cancer is possible because CaP is a sex hormone dependent cancer
(Smith, P. (1995), Cancer Surveys Vol. 23: Preventing Prostate
Cancer, Imper. Cancer Research Fund); that is, the growth of the
cancer is promoted by male hormones (e.g., androgens such as
testosterone and dihydrotestosterone). Removal of the testes
(castration) was for many years the standard method of preventing
secretion of male hormones by the gonads, to reduce growth of the
cancer. Currently, secretion of male hormones is suppressed by
chemical means by interfering with production of luteinizing
hormone (LH), which regulates synthesis of male hormones. Similar
considerations are applicable to other sex hormone-dependent
cancers, such as breast or ovarian cancer.
[0029] Beyond detection of elevated PSA level, other current
diagnostic methods for CaP are known to medical practitioners
skilled in the art and include rectal examination, transrectal
ultrasonography or magnetic resonance imaging (MRI), bone scanning,
X-rays, skeletal survey, intravenous pyelography, CAT-scan, and
biopsy (reviewed in Garnick, M. (1993), Annals of Internal
Medicine, 118:803-818; and Garnick, M. (1994), Scientific American,
270:72-81). These procedures are invasive, complex, costly, and
require highly trained personnel.
[0030] CaP stages are commonly evaluated according to a scale
divided as A, B, C and D. Tumors in stage A are microscopic; stage
A.sub.1 designates tumors confined to a relatively small area and
composed of well-differentiated tissue; stage A.sub.2 tumors are
more diffuse and less well differentiated; stage B tumors are large
enough to be felt during a rectal examination; and stage C prostate
cancers have spread throughout the gland and typically have pushed
past the borders of the prostate into surrounding structures. Stage
D tumors have metastasized, e.g., to lymph nodes, bone, or other
organs. Alternatively, tumors are also staged by the TNM staging
system, in which tumors are ranked on a scale of progressively
worsening disease from T1a to T4b (e.g., T1c tumors are
non-palpable and non-visible that were detected by elevated blood
levels of prostate specific antigen). Of tumors characterized as
being is stages A2, B, or C, 25% to 50% turn out, on further
testing, to be metastatic (Garnick, supra). Methods involving
procedures for removal or destruction of prostatic tumor tissue
usually are employed with non-metastasized cancers. For example,
radical prostatectomy preferably is used with stage A, B and some
stage C tumors (i.e., where the tumor growth has not extended
considerably beyond the borders of the prostate gland) as well as
stage T1c tumors. X-ray therapy (e.g., external or interstitial)
preferably is used with stage A, B or C tumors as well as T1c
tumors. Additional diagnostic tools might aid in distinguishing
cases suitable for various treatments.
[0031] In the present invention, detection of a pattern of enzymes
in a biological sample from a subject is used to facilitate
diagnosis and prognosis of TRAC by offering statistical
associations with particular conditions. The term "enzymes" is art
recognized and includes protein catalysts of chemical reactions.
The enzyme for purposes of this invention can be a whole intact
enzyme or portions or fragments thereof. The preferred embodiment
of the term enzymes as used herein, are naturally occurring enzymes
that catalytically degrade proteins, i.e. the enzymes known as
proteases or proteinases. By proteinase is meant a progressive
exopeptidase that digest proteins by removing amino acid residues
from either the N terminal or C terminal which reaction proceeds to
achieve significant degradation, or an endopeptidase which destroys
the amide bond between amino acid residues with varying degrees of
residue specificity. The term "protease" may also include the
highly specific amino acid peptidases that remove a single amino
acid from an N terminus or C terminus of a protein. Examples are
alanine aminopeptidase (EC 3.4.11.2) and leucine aminopeptidase (EC
3.4.11.1), which remove alanine or leucine, respectively, from the
amino terminus of a protein that may have alanine and leucine,
respectfully, at the amino terminus. Molecular weights of enzymes
of the invention are included in, but not limited to, molecular
weights in the range of approximately 20 kDa to approximately 200
kDa, more preferably 50 kDa to approximately 150 kDa.
[0032] The more preferred embodiment of the term protease
designates an enzyme which can actually cause digestion of a
protein yielding a product with a significantly lower molecular
weight than the substrate material. The term "enzyme" includes
polymorphic variants that are silent mutations naturally found
within the human population. The enzymes in the best embodiment of
the invention are proteases or proteinases, however there is no
intent to limit the invention to these enzymes. The term proteases
(and its equivalent term proteinases) is intended to include those
endopeptidases and progressive exopeptidases that are capable of
substantially reducing the molecular weight of the substrate and
destroying its biological function, especially if that biological
function of the substrate is to be a structural component of a
matrix barrier. Amino acid peptidases such as alanine
aminopeptidase and leucine aminopeptidase are also broadly included
among proteases, however do not share the property of significantly
reducing the molecular weight of the substrate protein.
[0033] Many thousands of proteases occur naturally, and each may
appear at different times of development and in different locations
in an organism. The invention herein features enzymes of the class
of the matrix metalloproteinases (MMPs, class EC 3.4.24). These
enzymes, which require a divalent cation for activity, are normally
expressed early in the development of the embryo, for example,
during hatching of an zygote from the zona pellucida, and again
during the process of attachment of the developing embryo to the
inside of the uterine wall. Enzyme activities such as
N-acetylglucosaminidase (EC 3.2.1.50) appear in urine in the case
of renal tubular damage, for example, due to diabetes (Carr, M.,
(1994), J. Urol. 151(2):442-445; Jones, A., et. al., (1995),
Annals. Clin. Biochem., 32:68-62). That these activities appear in
urine as a result of renal tubular damage is irrelevant to the
present invention as described herein.
[0034] The language "matrix-digesting enzyme" refers to an enzyme
capable of digesting or degrading a matrix, e.g., a mixture of
proteins and proteoglycans that comprise a layer in a tissue on
which certain types of cells are found. Matrix-digesting enzymes
are expressed during stages of normal embryogenesis, pregnancy and
other processes involving tissue remodelling. In addition, some of
these enzymes, for example some matrix metalloproteinases (MMPs),
degrade the large extracellular matrix proteins of the parenchymal
and vascular basement membranes that serve as mechanical barriers
to tumor cell migration. These MMPs are produced in certain cancers
and are associated with metastasis (Liotta, L. A., et al., (1991),
Cell, 64:327-336). Examples of MMPs are the type IV collagenases,
e.g., mmp-2 (gelatinase A. EC 3.4.24.24) and mmp-9 (gelatinase B,
3.4.24.35), and stromelysins (EC 3.4.24.17 and 3.4.24.22). Some
MMPs are specifically inhibited by molecules called tissue
inhibitors of metalloproteinases (TIMPs, Woessner, J. F., Jr.,
(1995), Ann. New York Acad. Sci., 732:11-21), which also may be
overproduced by tumor cells, however under certain conditions
enzyme activity is in molar excess over the TIMPs (Freeman, M. R.
et al., (1993), J. Urol., 149:659; Lu, X. et al., (1991), Cancer
Res., 51:6231-6235,; Kossakowska, A. E. et al., (1991), Blood,
77:2475-2481). In one embodiment of this invention, the inhibitors
of the enzyme (TIMPS, e.g., TIMP-1 or TIMP-2) may be the biological
markets detected in the biological sample (e.g., complexed or free
form) rather than the enzyme per se. The detection of the
inhibitors can be accomplished using art-recognized techniques.
Many of MMPs are translated as pro-enzymes, and may be found in a
variety of structures, with ranges of molecular weights including
smaller forms (45 kDa, 55 kDa, 62 kDa), and larger forms (72 kDa,
82 kDa, 92 kDa, and higher polymers such as 150 kDa and
greater).
[0035] The language "prostate cancer-associated enzyme" or
"prostate disorder-associated enzyme" is intended to include
enzymes whose detection in a biological sample of a subject would
facilitate diagnosis of prostate cancer or a prostate disorder.
Examples include MMPs in a range of approximately 20 kDa to 200
kDa, more preferably approximately 50 kDa to approximately 150 kDa,
e.g., more particularly approximately 72 kDa, 92 kDa, or 150 kDa.
Presentation of these sizes do not preclude additional enzymes of
larger or smaller sizes. In one embodiment of the invention, the
enzyme is not an enzyme of a size of approximately 72 kDa or
approximately 44 kDa. In addition to the MMPs, other types of
proteases may be produced and activated in some types of cancer,
for example, plasminogen may be expressed and activated by
proteolytic cleavage to the protease plasmin (EC 3.4.21.7).
[0036] The term "electrophoresis" is used to indicate any
separation system of molecules in an electric field, generally
using an inert support system such as paper, starch gel, or
preferably, polyacrylamide. The electrophoresis methods with
polyacrylamide gels and the sodium dodecyl sulfate denaturing
detergent are described in the Examples below. The protocols are
not intended to exclude equivalent procedures known to the skilled
artisan. Other SDS polyacrylamide procedures, known to the skilled
artisan, may be used, e.g., a single polyacrylamide concentration
such as 10%, may be substituted for the gradient in the separation
gel. The physical support for the electrophoretic matrix may be
capillary tubes rather than glass plates. Details of several
SDS-polyacrylamide gel electrophoresis systems are described in
many review articles and biotechnology manuals (e.g., Maniatis, T.,
Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y.). The method is not limited
to use of SDS and other detergents. Further, electrophoresis in the
absence of detergents may be employed. Proteins may be separated
under non-denaturing conditions, for example in the presence of
urea on a polyacrylamide matrix (Maniatis, supra), or by charge,
for example by the procedure of iso-electric focussing.
[0037] In using an electrophoretic technique for separation of
enzymes, the electrophoretogram may be developed as a zymogram. The
term "zymography" is meant here to include any separations system
utilizing a chemically inert separating or support matrix, that
allows detection of an enzyme following electrophoresis, by
exposing the matrix of the separations system to conditions that
allow enzyme activity and subsequent detection. More narrowly, the
term zymography designates incorporation of an appropriate
substrate for the enzyme of interest into the inert matrix, such
that exposing the matrix to the conditions of activity after the
electrophoresis stop yields a system to visualize the precise
location, and hence the mobility, of the active enzyme. By
techniques well-known to the skilled artisan, the molecular weights
of proteins are calculated based on mobilities derived from
positions on a zymogram. Such techniques include comparison with
molecular weight standards, the mobilities of which are determined
from general protein stains or from pre-stains specific to those
standards, and comparison with positive controls of purified
isolated enzymes of interest, which are visualized by the technique
of the zymogram, i.e., enzyme activity.
[0038] In particular, substrates for detection of proteases by
zymography are included in the electrophoresis matrix. For type IV
collagenases, the natural substrate is a type IV collagen and
gelatin, a type I collagen derivative, is used for the zymography
substrate in the Examples presented herein. However other proteins
that are suitable for detection of further proteases of interest in
TRAC diagnosis, for example, include fibronectin; vitronectin;
collagens of types I through III and V through XII; procollagens;
elastin; laminin; plasmin; plasminogen; entactin; nidogen;
syndecan; tenascin; and sulfated proteoglycans substituted with
such saccharides as hyaluronic acid, chondroitin-6-sulfate,
condroitin-4-sulfate, heparan sulfate, keratan'sulfate, and
dermatan sulfate and heparin. Further, convenient inexpensive
substrate proteins such as casein, which may not be the natural
target of a protease of interest, but are technically appropriate,
are included as suitable substrate components of the zymography
techniques of the present invention. Chemically synthesized
mimetics of naturally occurring protein substrates are also
potential zymography substrates, and may even be designed to have
favorable properties, such chromogenic or fluorogenic ability to
produce a color or fluorescent change upon enzymatic cleavage.
[0039] Zymography may be adapted to detection of a protease
inhibitor in the biological sample. Since a variety of natural MMP
inhibitors are elaborated, such as TIMP-1 and TIMP-2, and are found
to be deregulated during TRAC situations, the present invention
includes detection of enzyme inhibitors as well as the enzymes of
tissue remodelling. Thus for example, a "reporter enzyme" for which
enzyme an inhibitory activity is being measured, may be incubated
with each biological sample obtained by subjects and patients, in
one or more quantities corresponding to one or more aliquots of
sample, prior to electrophoresis. This enzyme is omitted from one
aliquot of the biological sample. The inhibitory presence in the
sample is detected as disappearance or decrease of the reporter
enzyme band from the developed zymogram. Alternatively, functional
enzyme activity assays which include in the reaction mix a known
level of active enzyme, to which is added aliquots of experimental
samples with putative inhibitory activity, can detect the presence
of inhibitors.
[0040] Further, the enzymes of tissue remodelling extend to enzyme
activities beyond those of proteolytic activity. For example,
enzymes that are substituted with residues such as glycosyl,
phosphate, sulfate, lipids and nucleotide residues (e.g. adenyl)
are well-known to those skilled in the art. These residues are in
turn added or removed by other enzymes, e.g., glycosidases,
kinases, phosphatases, adenyl transferases, etc. Convenient
detection methods for the presence of such activities for TRAC
diagnosis and prognosis are readily developed by those with skill
in the art, and are intended to comprise part of the invention
here.
[0041] If activities are found in urine associated with renal
damage, these may be detected by the methods described here, in
which case positive data obtained with the methods of the present
invention must be evaluated by including renal damage as a
causative condition. Overlap between renal damage and TRAC
diagnoses of course exist, e.g., renal cancer, renal tubule
obstruction, renal ulcers, etc. The utility of the present
invention is not construed as limited by the possibilities of an
overlap, nor limited by knowledge of such conditions.
[0042] The zymogram as described in the Examples herein is
developed by use of a general stain for protein, in this case,
Coomassie Blue dye. The development is possible with general
protein stains, e.g., Amido Black dye, and SYPRO Orange stain
(Biorad Laboratories, Hercules, Calif. 94537). Further, enzyme
activity may be detected by additional techniques beyond that of a
clear zone of digestion in a stained matrix, for example, by
absence of areas of radioactivity with a radio-labelled substrate,
by change in mobility of a radio-labelled substrate, or by absence
of or change in mobility of bands of fluorescence or color
development with use of fluorogenic or chromogenic substrates,
respectfully.
[0043] Quantitative densitometry can be performed with zymograms by
placing the gel directly on an activated plate of a Molecular
Dynamics phosphorimager (Molecular Dynamics, 928 East Arques Ave.,
Sunnyvale, Calif. 94086), or with a Datacopy G8 plate scanner
attached to a MacIntosh computer equipped with an 8-bit videocard
and McImage (Xerox Imaging Systems). Background measurements, areas
of the gel separate from sample lanes, can similarly be scanned,
and values subtracted from the readings for enzyme activities.
[0044] Another electrophoretically-based technique for analysis of
a biological sample for presence of specific proteins is an
affinity-based mobility alteration system (Lander, A., (1991),
Proc. Natl. Acad. Sci. U.S., 88(7):2768-2772). An MMP or other type
of enzyme of interest might be detected, for example, by inclusion
of a substrate analog that binds essentially irreversibly to the
enzyme, hence decreasing the mobility. The affinity material is
present during electrophoresis, and is incorporated into the
matrix, so that detection of the enzyme of interest occurs as a
result of alteration of mobility in contrast to mobility in the
absence of the material. Yet another technique of electrophoretic
protein separation is based on the innate charge of a protein as a
function of the pH of the buffer, so that for any protein species,
there exists a pH at which that protein will not migrate in an
electric field, or the isoelectric point, designated pI. Proteins
of a biological sample, such as a urine sample, may be separated by
isoelectric focussing, then developed by assaying for enzymatic
activity for example by transfer to material with substrate, i.e.,
zymography. Electrophoresis is often used as the basis of
immunological detections, in which the separation step is followed
by physical or electrophoretic transfer of proteins to an inert
support such as paper or nylon (known as a "blot"), and the blotted
pattern of proteins may be detected by use of a specific primary
binding (Western blot) by an antibody followed by development of
bound antibodies by secondary antibodies bound to a detecting
enzyme such as horse radish peroxidase. Additional immunological
detection systems for TRAC enzymes are now described in detail
below.
[0045] The term "antibody" as used herein is intended to include
fragments thereof which are also specifically reactive with one of
the components in the methods and kits of the invention. Antibodies
can be fragmented using conventional techniques and the fragments
screened for utility in the same manner as described above for
whole antibodies. For example, F(ab).sub.2 fragments can be
generated by treating an antibody with pepsin. The resulting
F(ab).sub.2 fragment can be treated to reduce disulfide bridges to
produce Fab fragments. The term "antibody" is further intended to
include single chain, bispecific and chimeric molecules. The term
"antibody" includes possible use both of monoclonal and polyclonal
antibodies (Ab) directed against a target, according to the
requirements of the application.
[0046] Polyclonal antibodies can be obtained by immunizing animals,
for example rabbits or goats, with a purified form of the antigen
of interest, or a fragment of the antigen containing at least one
antigenic site. Conditions for obtaining optimal immunization of
the animal, such as use of a particular immunization schedule, and
using adjuvants e.g. Freund's adjuvant, or immunogenic substituents
covalently attached to the antigen, e.g. keyhole limpet hemocyanin,
to enhance the yield of antibody titers in serum, are well-known to
those in the art. Monoclonal antibodies are prepared by procedures
well-known to the skilled artisan, involving obtaining clones of
antibody-producing lymphocyte, i.e. cell lines derived from single
cell line isolates, from an animal, e.g. a mouse, immunized with an
antigen or antigen fragment containing a minimal number of
antigenic determinants, and fusing said clone with a myeloma cell
line to produce an immortalized high-yielding cell line. Many
monoclonal and polyclonal antibody preparations are commercially
available, and commercial service companies that offer expertise in
purifying antigens, immunizing animals, maintaining and bleeding
the animals, purifying sera and IgG fractions, or for selecting and
fusing monoclonal antibody producing cell lines, are available.
[0047] Specific high affinity binding proteins, that can be used in
place of antibodies, can be made according to methods known to
those in the art. For example, proteins that bind specific DNA
sequences may be engineered (Ladner, R. C., et. al., U.S. Pat. No.
5,096,815), and proteins that bind a variety of other targets,
especially protein targets (Ladner, R. C., et. al., U.S. Pat. No.
5,233,409; Ladner, R. C., et.al., U.S. Pat. No. 5,403,484) may be
engineered and used in the present invention for covalent linkage
to a chelator molecule, so that a complex with a radionuclide may
be formed under mild conditions. Antibodies and binding proteins
can be incorporated into large scale diagnostic or assay protocols
that require immobilizing the compositions of the present invention
onto surfaces, for example in multi-well plate assays, or on beads
for column purifications.
[0048] General techniques to be used in performing various
immunoassays are known to those of ordinary skill in the art.
Moreover, a general description of these procedures is provided in
U.S. Pat. No. 5,051,361 which is incorporated herein by reference,
and by procedures known to the skilled artisan, and described in
manuals of the art (Ishikawa, E., et. al. (1988), Enzyme
Immunoassay Igaku-shoin, Tokyo, N.Y.; Hallow, E. and D. Lane,
Antibodies: A Laboratory Manual, CSH Press, NY). Examples if
several immunoassays are given discussed here.
[0049] Radioimmunoassays (RIA) utilizing radioactively labeled
ligands, for example, antigen directly labeled with .sup.3H, or
.sup.14C, or .sup.125I, measure presence of MMP's as antigenic
material. A fixed quantity of labeled MMP antigen competes with
unlabeled antigen from the sample for a limited number of antibody
binding sites. After the bound complex of labeled antigen-antibody
is separated from the unbound (free) antigen, the radioactivity in
the bound fraction, or free fraction, or both, is determined in an
appropriate radiation counter. The concentration of bound labeled
antigen is inversely proportional to the concentration of unlabeled
antigen present in the sample. The antibody to MMP can be in
solution, and separation of free and bound antigen MMP can be
accomplished using agents such as charcoal, or a second antibody
specific for the animal species whose immunoglobulin contains the
antibody to MMP. Alternatively, antibody to MMP can be attached to
the surface of an insoluble material, which in this case,
separation of bound and free MMP is performed by appropriate
washing.
[0050] Immunoradiometric assays (IRMA) are immunoassays in which
the antibody reagent is radioactively labeled. An IRMA requires the
production of a multivalent MMP conjugate, by techniques such as
conjugation to a protein e.g., rabbit serum albumin (RSA). The
multivalent MMP conjugate must have at least 2 MMP residues per
molecule and the MMP residues must be of sufficient distance apart
to allow binding by at least two antibodies to the MMP. For
example, in an IRMA the multivalent MMP conjugate can be attached
to a solid surface such as a plastic sphere. Unlabeled "sample" MMP
and antibody to MMP which is radioactively labeled are added to a
test tube containing the multivalent MMP conjugate coated sphere.
The MMP in the sample competes with the multivalent MMP conjugate
for MMP antibody binding sites. After an appropriate incubation
period, the unbound reactants are removed by washing and the amount
of radioactivity on the solid phase is determined. The amount of
bound radioactive antibody is inversely proportional to the
concentration of MMP in the sample.
[0051] Other preferred immunoassay techniques use enzyme labels
such as horseradish peroxidase, alkaline phosphatase, luciferase,
urease, and .beta.-galactosidase. For example, MMP's conjugated to
horseradish peroxidase compete with free sample MMP's for a limited
number of antibody combining sites present on antibodies to MMP
attached to a solid surface such as a microtiter plate. The MMP
antibodies may be attached to the microtiter plate directly, or
indirectly, by first coating the microtiter plate with multivalent
MMP conjugates (coating antigens) prepared for example by
conjugating MMP with serum proteins such as rabbit serum albumin
(RSA). After separation of the bound labeled MMP from the unbound
labeled MMP, the enzyme activity in the bound fraction is
determined colorimetrically, for example by a multi-well microtiter
plate reader, at a fixed period of time after the addition of
horseradish peroxidase chromogenic substrate.
[0052] Alternatively, the antibody, attached to a surface such as a
microtiter plate or polystyrene bead, is incubated with an aliquot
of the biological sample. MMP present in the fluid will be bound by
the antibody in a manner dependent upon the concentration of MMP
and the association constant between the two. After washing, the
antibody/MMP complex is incubated with a second antibody specific
for a different epitope on MMP distal enough from the MMP-specific
antibody binding site such that steric hindrance in binding of two
antibodies simultaneously to MMP may be accomplished. For example,
the second antibody may be specific for a portion of the proenzyme
sequence. The second antibody can be labeled in a manner suitable
for detection, such as by radioisotope, a fluorescent compound or a
covalently linked enzyme. The amount of labeled secondary antibody
bound after washing away unbound secondary antibody is proportional
to the amount of MMP present in the biological sample.
[0053] The above examples of preferred immunoassays describe the
use of radioactively and enzymatically labeled tracers. Assays also
may include use of fluorescent materials such as fluorescein and
analogs thereof, 5-dimethylaminonaphthalene-1-sulfonyl derivatives,
rhodamine and analogs thereof, coumarin analogs, and
phycobiliproteins such as allophycocyanin and R-phycoerythrin;
phosphorescent materials such as erythrosin and europium;
luminescent materials such as luminol and luciferin; and sols such
as gold and organic dyes. In one embodiment of the present
invention, the biological sample is treated to remove low molecular
weight contaminants.
[0054] In one embodiment of the present invention, the biological
sample is treated to remove low molecular weight contaminants, for
example, by dialysis. By the term "dialysis" this invention
includes any technique of separating the enzymes in the sample from
low molecular weight contaminants. The Examples use Spectra/Por
membrane dialysis tubing with a molecular weight cut-off (MWCO) of
3,500, however other products with different MWCO levels are
functionally equivalent. Other products include hollow fiber
concentration systems consisting of regenerated cellulose fibers
(with MWCO of 6,000 or 9,000) for larger volumes; a multiple
dialyzer apparatus with a sample size for one to 5 ml; and multiple
microdialyzer apparatus, convenient for samples in plates with 96
wells and MWCOs at 5,000, 8,000 and 10,000, for example. These
apparatuses are available from PGC Scientific, Gaithersburg, Md.,
20898. Those with skill in the art will appreciate the utility of
multiple dialysis units, and especially suitable for kits for
reference lab and clinic usage. Other equivalent techniques include
passage through a column holding a resin or mixture of resins
suitable to removal of low molecular weight materials. Resins such
as BioGel (BioRad, Hercules, Calif.) and Sepharose (Pharmacia,
Piscataway, N.J.) and others are well-known to the skilled artisan.
The technique of dialysis, or equivalent techniques with the same
function, are intended to remove low molecular weight contaminants
from the biological fluids. While not an essential component of the
present invention, the step of removal of such contaminants
facilitates detection of the disorder-associated enzymes in the
biological samples.
[0055] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference.
EXAMPLES
[0056] The following methodology described in the Materials and
Methods section was used throughout these Examples, set forth
below.
Material and Methods
[0057] Patient Groups
[0058] Urine samples were obtained from subjects with
clinically-determined cancers of the following types: prostate
cancer (13 subjects, Table 1), metastatic cancer (9 subjects, Table
2), and other non-metastatic malignancies in a variety of organs
(11 subjects, Table 3). Further, urine samples were obtained from
subjects with no history of cancer (13 subjects, Table 4), with
benign prostatic hyperplasia. (8 subjects Table 5), with no
evidence of disease (15 subjects, Table 6), and under treatment by
hormonal suppression (4 subjects, Table 7). Urine samples of breast
cancer patients were tested for the presence of menstrual blood
using Ames Multistix 7 reagent strips (Miles, Elkhardt, Ind.), and
those containing blood were not analyzed further. These specimens
were analyzed by gelatin zymography, and the results were recorded
as positive for each protein band with gelatinase activity observed
in the lane corresponding to that urine sample.
[0059] Preparation of Samples
[0060] Urine samples were kept frozen until assay, thawed overnight
at 4 C, and a 10 ml aliquot was dialyzed against double-distilled
water in 45 mm dialysis tubing (Spectra/Por membrane MWCO: 3,500,
Spectrum, Houston, Tex.). Following dialysis, urine samples were
centrifuged at 4,000 rpm for 5 min at 4 C, the supernatant was
taken and stored at 4 C prior to analysis. Analysis of urine for
enzyme activity was by zymography, comprising gel electrophoresis
performed in the presence of enzyme substrate followed by in situ
digestion.
[0061] Electrophoresis
[0062] Polyacrylamide gel electrophoresis in the presence of sodium
dodecyl sulfate (SDS) and 0.1 % (w/v) gelatin was used in the
Examples here. A sample comprising 30 microliters of dialyzed urine
was mixed with 15 microliters of sample buffer consisting of 4%
SDS, 0.15 M Tris pH 6.8, 20% (v/v) glycerol, and 0.5% (w/v)
bromphenol blue. Samples were applied to slots in a 4% stacking gel
above a 10% polyacrylamide separating gel on a mini-gel slab gel
apparatus (Mini-Protein II, Bio-Rad).
[0063] Zymography
[0064] Following electrophoresis, gels were incubated for 30 min in
2.5% Triton X-100 (v/v) to remove SDS, rinsed, and incubated
overnight at 37 C in substrate buffer (0.05 M Tris, pH 8.5, 5 mM
CaCl.sub.2, 0.02% sodium azide). To visualize bands of active
enzyme, gels were stained for protein in 0.5% (w/v) Coomassie Blue
R-250 in acetic acid: isopropanol: water (1:3:6), then destained in
acetic acid ethanol water (1:3:6). Enzyme activity appeared as
clear bands in the dark background, corresponding to the lane for
each sample. The electrophoretic mobility of each clear band was
determined by correlation with molecular weight protein standards
and positive controls (purified mmp-2 and mmp-9 proteins). The
molecular weight of each band of active enzyme was recorded
according to the following criteria: greater than 150 kDa, 92 kDa,
72 kDa, and other molecular weights (for example, 100 kDa and 20
kDa). The identity of these MMPs was confirmed by western blot
analysis using anti-MMP antibodies (Oncogene Sciences, Cambridge,
Mass.).
[0065] Gels were analyzed by the double blind method, in which
reading and scoring the gel patterns was performed by the
experimenter without knowledge of the identity of each sample. A
band of enzyme activity is indicated by a "yes" in the Tables in
the Examples below. Patients with several readings taken from time
to time because of possible disease progression are here included
only in the grouping of most recent diagnosis, i.e., each coded
subject is represented in one Table only.
[0066] To verify that enzyme activities observed by zymography
detected were metal-dependent proteases, samples were subjected
also to incubation in substrate buffer in the presence of
1,10-phenanthroline, an MMP inhibitor.
Normal Controls
[0067] Samples of urine from 13 young healthy male subjects
revealed 8 positives of 52 possible metalloproteinase bands (15%,
Table 1). Thus, 4 of 13 (31%) healthy subjects had detectable
gelatinase activity in urine. Analysis of the distribution of the
enzyme activity shows that none of the urine samples contained
enzyme activity of 72 kDa size.
1TABLE 1 Zymograms of Normal Subjects(13 subjects) enzyme pattern
in urine Code >150 kDa 92 kDa 72 kDa other MMPs No-1 yes yes no
no No-2 no no no no No-3 no no no no No-4 no no no no No-5 no no no
no No-6 yes yes no no No-7 no yes no no No-8 yes yes no yes No-9 no
no no no No-10 no no no no No-11 no no no no No-12 no no no no
No-13 no no no no Subjects No-1 through No-13 are normal, and have
no medical history of cancer.
Example 1
Enzyme Activity in Urine of Prostate Cancer Patients and Other
Cancer Patients
[0068] Analysis of urine of CaP patients showed gelatinase in 12 of
the 13 samples (Table 2) Thus 92% of urine from CaP patients
contained a band of 92 kDa or higher molecular weight, and 48% of
the 52 possible zymogram activity categories are positive for the
CaP group. These frequencies are over 3-fold higher than comparable
findings for the urines from the normal controls.
[0069] Urine samples from 5 out of 11 of patients with other types
of cancer showed metalloproteinase activity (Table 3). Enzymes were
found in urine from 3 out of 5 bladder cancer patients, and in
urine from one out of 2 patients with renal cancer, and one out of
2 patients with lymphoma.
Example 2
Enzyme Activity in Urine of Metastatic Cancer Patients
[0070] In the MC patient group, urine samples of all 8 patients
displayed metalloproteinase activity (Table 4). Of the 32 possible
enzyme band categories recorded, 66% were positive for patients
with metastatic cancer. Further, the urine of all MC patients
contained either enzyme of 92 kDa size, enzyme of molecular weight
greater than 150 kDa, or both.
2TABLE 2 Zymograms of Subjects with Prostate Cancer (13 subjects)
enzyme pattern in urine Code >150 kDa 92 kDa 72 kDa other MMPs
CaP-1 yes yes yes no CaP-2 yes yes yes no CaP-3 yes yes yes no
CaP-4 yes yes yes no CaP-5 no yes no no CaP-6 no yes no no CaP-7
yes yes no no CaP-8 no yes no no CaP-9 no no no no CaP-10 no yes no
no CaP-11 yes yes no no CaP-12 yes yes yes no CaP-13 yes yes no no
Subjects CaP-1 through CaP-13 are prostate cancer patients.
[0071]
3TABLE 3 Zymograms of Subjects with Other Cancers. Non-Metastatic
(11 subjects) enzyme pattern in urine other Code >150 kDa 92 kDa
72 kDa MMPs cancer CaB-1 no no no no bladder CaB-2 no no no no
bladder CaB-3 yes yes no no bladder CaB-4 yes yes yes no bladder
CaB-5 yes yes no yes bladder CaR-1 no no no no renal CaR-2 yes yes
yes yes renal CaLy-1 no no no no lymphoma CaLy-2 yes yes no no
lymphoma CaT-1 no no no no testis CaPh-1 no no no no
pheochromocytoma Five subjects with bladder cancer are indicated
CaB-1 through -5. CaR-1 and -2 are subjects with renal cancer.
CaLy-1 and -2 are subjects with lymphoma, CaT-1 is a testicular
cancer patient, and CaPH-1 has pheochromocytoma.
[0072]
4TABLE 4 Zymograms of Subjects with Metastatic Cancer (8 subjects)
enzyme pattern in urine Code >150 kDa 92 kDa 72 kDa other MMPs
Meta-1 yes yes yes no Meta-2 yes yes yes yes Meta-3 yes yes no no
Meta-4 yes yes no no Meta-5 yes yes no yes Meta-6 no yes no no
Meta-7 yes no no yes Meta-8 yes yes yes yes Subjects MC-1 through
-8 are metastatic cancer patients.
[0073]
5TABLE 5 Zymograms of Subjects with Benign Prostatic Hyperplasia (8
subjects) enzyme pattern in urine Code >150 kDa 92 kDa 72 kDa
other MMPs BPH-1 yes yes no no BPH-2 yes yes no no BPH-3 no yes no
no BPH-4 no yes no no BPH-5 no yes no no BPH-6 no no no no BPH-7 no
no np no BPH-8 yes yes no yes Subjects indicated BPH-1 through -8
have benign prostatic hyperplasia.
Example 3
Enzyme Activity in Urine of Subjects with Benign Prostatic
Hypertrophy
[0074] Eight subjects with BPH were assayed for metalloproteinase
content of urine (Table 5). Of the 32 possible enzyme pattern
observations, 10 were positive (31%), and 6 of these 8 patients
(75%) had urine containing one or more bands of activity, a
frequency higher than that of the normal subjects and those with no
evidence of disease. None of the BPH subjects' urines (0%) showed
72 kDa metalloproteinase band. The MMP pattern of the BPH subjects
as a function of time can be used to facilitate prognosis of
patients likely to develop problematic BPH or other conditions.
Example 4
Enzyme Activity in Urine of Subjects with No Evidence of
Disease
[0075] Fifteen subjects with previous medical histories of cancer,
with no recent evidence of disease, and under regular clinical
observation, were assayed for metalloproteinase content of urine.
Three (20%) of the subjects' urine were found to contain enzyme
(Table 6). None of these specimens contained 72 kDa
metalloproteinase, and 12% (7 of 60 possible band recordings) of
the possible band data from this group were positive. Most of these
patients were at one time under treatment for cancer, and have
exhibited no symptoms in the recent past and at the time of
collection of urine samples. The frequencies of positive results
were consistent with that of normal subjects.
Example 5
Enzyme Activity in Urine of Subjects under Hormonal Suppression
[0076] Table 7 shows data on metalloproteinase activity pattern in
the urine specimens of 4 patients diagnosed with prostate cancer in
the past, and currently under treatment by hormonal suppression. Of
the 16 possible data entries, one is positive (6%), and thus one of
the 4 patients has MMP activity in the urine (25%). The urine
enzyme patterns of patients under hormonal suppression, which is
used to prevent or delay return of prostate cancer, shows reduced
frequency of positive metalloproteinase bands in urine compared to
the cancer groups.
6TABLE 6 Zymograms of Subjects with No Evidence of Disease 15
subjects) enzyme pattern in urine Code >150 kDa 92 kDa 72 kDa
other MMPs NeD-1 no no no no NeD-2 no no no no NeD-3 no no no no
NeD-4 yes yes no no NeD-5 no no no no NeD-6 no no no no NeD-7 no no
no no NeD-8 no no no no NeD-9 no no no no NeD-10 no no no no NeD-11
yes yes no no NeD-12 no no no no NeD-13 no no no no NeD-14 yes yes
no yes NeD-15 no no no no NeD-1 through -15 are subjects with prior
history of cancer and with no evidence of disease in recent medical
history and at the time of the urine sample.
[0077]
7TABLE 7 Zymograms of Hormonally Suppressed Subjects (4 subjects)
enzyme pattern in urine Code >150 kDa 92 kDa 72 kDa other MMPs
HS-1 no no no no HS-2 no no no no HS-3 no no no no HS-4 no yes no
no HS-1, -2, -3 and -4 are subjects with a history of prostate
cancer, under treatment by hormonal suppression.
[0078]
8TABLE 8 Summary of Urine Metalloproteinase Activities in 4
Molecular Weight Categories for All Subjects diagnostic category
number positive/total percent positive normal (no cancer history)
8/52 15% prostate cancer, organ confined 25/52 48% other cancers
(non-metastatic) 14/43 33% metastatic cancer 21/32 66% benign
prostatic hyperplasia 10/32 31% no evidence of disease 7/60 12%
hormonal suppression 1/16 6%
[0079]
9TABLE 9 Summary of 92 kDa Metalloproteinase in Urine disease
status number positive/total percent positive normal (no cancer
history) 4/13 31% prostate cancer, organ confined 12/13 92% other
cancers (non-metastatic) 5/11 46% metastatic cancer 7/8 88% benign
prostatic hyperplasia 6/8 75% no evidence of disease 3/15 20%
hormonal suppression 1/4 25%
[0080] These data, for subjects in Examples 1-5 above, are
summarized in Table 8 by diagnostic category of the subjects. Table
8 shows that 48% of the categories of metalloproteinase molecular
species from urine of subjects with organ-confined prostate cancer,
and 66% of those with metastatic cancer, were positive bands of
enzyme activity. Controls of urine from normal subjects, from
subjects with no evidence of disease, and from hormonally
suppressed subjects with a history of prostate cancer, were 15%,
12% and 6%, respectively.
10TABLE 10 Summary of 72 kDa Metalloproteinase Activity in Urine
disease status number positive/total percent positive normal (no
cancer history) 0/13 0% prostate cancer, organ confined 5/13 38%
other cancers (non-metastatic) 2/11 18% metastatic cancer 3/8 38%
benign prostatic hyperplasia 0/8 0% no evidence of disease 0/15 0%
hormonal suppression 0/4 0%
[0081] The presence of enzyme activity of 72 kDa MMP in urine
(Table 10) is uniquely found in urine from the cancer groups: 38%
of subjects with organ-confined prostate cancer (5 of 13) contained
this metalloproteinase, compared to none of the subjects in any of
the non-cancer groups. The data show that the pattern of presence
of particular of full-length metalloproteinase mmp-2 and mmp-9
proenzymes, and of high molecular weight MMPs (greater than 150
kDa), are diagnostic and prognostic tools.
Example 6
Statistical Analysis of Urine MMP Enzyme Patterns in Urine as a
Cancer Marker
[0082] The urinary MMP pattern data were submitted for statistical
analysis using logistic regression, in which prostate and other
non-metastatic cancer patients versus the combination of the normal
and no evidence of disease (NeD) group is considered the binary
outcome.
[0083] Comparing normal/NeD versus Cancer, the univariate results
of the analysis, for greater than 150 kDa, 92 kDa and 72 kDa MMPs
were that a significantly higher proportion of each of these MMP
categories was detected in patients with cancer. The "other MMP"
category was not significantly different between the cancer group
and normal/NeD, i.e., not a significant predictor. Further, the
odds are roughly 5 times higher for cancer patients to have MMP of
greater than 150 kDa molecular weight detected in the urine
compared to normal/NeD (the odds ratio equals 5.38, with a 95%
confidence interval of 1.80 to 16.12, likelihood ratio chi-square
test is 9.80, probability equals 0.002). The odds of detecting 92
kDa MMP are 7 times greater for the cancer patient group compared
to normal/NeD (the odds ratio equals 7.09, with a 95% confidence
interval of 4.53 to 40.67, likelihood ratio chi-square test is
13.57, probability less than 0.001). The odds of detecting 72 kDa
are estimated to be infinitely higher for those patients with
cancer (likelihood ratio chi-square test is 14.07, probability less
than 0.001) than the normal/NeD group.
[0084] The multivariate analysis establishes the most important MMP
markers and controls. The analysis indicates that 92 kDa and 72 kDa
MMPs are both significant independent predictors of cancer
(probability equals 0.01 for each). An estimate of the probability
of cancer for combinations of two independent multivariate
predictors is shown in Table 11. Subjects with urinary 72 kDa MMP
or with both 92 kDa and 72 kDa MMPs have very high probabilities of
having cancer, according to this analysis.
[0085] The probabilities of cancer predicted for eight possible
combinations of the three univariate predictors are shown in Table
12.
Example 7
Statistical Analysis of Urine MMP Enzyme Pattern as Metastatic
Cancer Markers
[0086] Statistical methods were used to compare the urinary MMP
patterns of subjects from the normal/NeD group versus patients with
metastasized cancers (MC). Ninety-five percent confidence limits
were derived using Pratt's method (Blyth, C. R., 1986, J.Am. Stat.
Assoc. 81: 843-855). For univariate analysis, the results indicate
that patients with MC are more likely to have each MMP present in
urine compared to normal/NeD. Patients with MC were nearly 13 times
more likely to have MMP of greater than 150 molecular weight
present in urine than normal/NeD (probability equals 0.002), and 10
times more likely to have 92 kDa MMP (probability equals 0.005) and
72 kDa MMP (probability equals 0.002). A higher proportion of
patients with MC compared to subjects in the normal/NeD group had
other urinary MMPs (probability equals 0.014).
[0087] Using multivariate analysis, the results for MC are that the
92 kDa and 72 kDa MMPs were significant independent predictors
(probability less than 0.05 for each). Modeling the estimated
probabilities of MC based on the 4 variant combinations of 92 kDa
and 72 kDa detected in the urine yielded the data in Table 13.
11TABLE 11 Probability of Cancer Predicted from the Combination of
Two Independent Multivariate Predictors, 92 kDa and 72 kDa MMP in
Urine. 92 kDa 72 kDa Probability of Cancer (%) - - 34.38 + - 68.18
- + 99.96 + + 99.99
[0088]
12TABLE 12 Probability of Cancer Predicted from Urine MMP Pattern
Combination of 3 Univariate Predictors. 150 kDa 92 kDa 72 kDa
Probability of Cancer (%) -- -- -- 34.53 -- -- + 99.99 -- + --
71.62 + -- -- 29.69 -- + + 99.99 + + -- 66.89 + -- + 99.95 + + +
99.99
[0089]
13TABLE 13 Probability of Metastatic Cancer Predicted from Urine
MMP Pattern of Two Univariate Predictors. 92 kDa 72 kDa Probability
of MC (%) -- -- 8.69 + -- 36.36 -- + 99.94 + + 99.99
[0090]
14TABLE 14 Gelatinase Profile. Cancer Type Controls
Metalloproteinases Prostate Renal Bladder Breast Other Metastatic
NeD Normal % with MMP 75 40 80 100 64 90 11 23 hMW 57 30 70 100 64
86 5 18 92 kDa 64 30 70 100 64 81 11 23 72 kDa 39 30 30 10 36 29 0
0 Creatinine (mg/dl): 131 (.+-.70*) 134 (.+-.78.degree.) 91
(.+-.52) 85 (.+-.42) 69 (.+-.38) 97 (.+-.60) 90 (.+-.47) 188
(.+-.76) *.+-.standard deviation; hMW = high molecular weight MMP
class migration at greater than or equal to 150 kDa.
[0091]
15TABLE 15 Statistical Performance Characteristics for Urine MMP
Markers* LR marker sensitivity (95% CI) specificity (95% CI)
positive Metastatic Cancers hMW 81.0 (58.1, 94.6) 87.8 (73.8, 95.9)
3.4 92 kDa 76.2 (52.8, 91.8) 82.9 (67.9, 92.9) 2.3 72 kDa 28.6
(11.2, 52.2) 100.0 (91.4, 100.0) ND hMW/72 kDa 81.0 (58.1, 94.6)
87.8 (73.8, 95.9) 3.4 Organ-Confined Cancers hMW 53.2 (38.1, 67.9)
87.8 (73.8, 95.9) 5.0 92 kDa 59.6 (44.3, 73.6) 82.9 (67.9, 92.9)
4.0 72 kDa 34.0 (20.9, 49.3) 100.0 (91.4, 100.0) ND 92/72 66.0
(50.7, 79.1) 82.9 (67.9, 92.9 4.4 All Cancers hMW 61.8 (49.2, 73.3)
87.8 (73.8, 95.9) 8.4 92 kDa 64.7 (52.2, 75.9) 82.9 (67.9, 92.9)
6.3 72 kDa 67.6 (55.2, 78.5) 100.0 (91.4, 100.0) ND 92/72 70.6
(58.3, 81.0) 82.9 (67.9, 92.9) 6.9 *These values are based on the
ability of each MMP to discriminate between the Normal+NeD samples
(n = 41) versus metastatic cancers (n = 21), organ confined cancers
(n = 47) and all cancers (n = 68). CI = confidence interval;
positive LR positive likelihood ratio (ratio of true positives to
false positives); ND = not defined because there were no false
positives. hMW = high molecular weight MMP class migrating at
greater than or equal to 150 kDa species. # The results of the
analysis of the combination of the 92 kDa and 72 kDa species, and
of the hMW and the 72 kDa species are given when these markers were
shown to be multivariate predictors in the logistic regression
analyses.
Example 8
Disease Progression in Cancer Patients and Change in Enzyme
Pattern
[0092] Patient Meta-4 (Table 4) was originally diagnosed with
organ-confined CaP, and was subsequently diagnosed with metastasis,
thus the data for this patient appear only in Table 4. Further,
urine from patient Meta-6 which displayed 92 kDa MMP as shown, had
been assayed 2 months prior to the data for that patient in Table
4, and was negative at that time for all MMP. These individual case
histories show the diagnostic and prognostic value for cancer
progression of the urine MMP pattern assay. These data showed that
there was a high correlation (greater than 99%) of the presence of
72 kDa MMP in urine and presence of cancer.
Example 9
Additional Patient Data
[0093] A total of 68 cancer patient urine samples have been
collected and analyzed for MMPs by the methods here. These include
28 patients with prostate cancer, 10 with renal cancer, 10 with
bladder cancer, 9 with breast cancer, and 11 with other cancer
(ovarian, lung, endometrial/cervical, testicular, lelomyosarcoma,
adrenal pheochromocytoma, transitional cell carcinoma of kidney and
lymphoma). These samples from patients with organ-confined cancers
were compared to those from 19 patients with metastatic cancer, 19
former cancer patients with no evidence of disease, and 22 normal
volunteers.
[0094] MMPs detected in the urine of patients with organ-confined
disease and with metastatic cancer were compared to the normal/no
evidence of disease control group. Sensitivity and specificity were
calculated using standard formulas and expressed as percentages.
The likelihood ratio was determined as the fraction of true
positives divided by the fraction of false positives
(sensitivity/100-specificity) to provide an indicator of the
discriminating power for each MMP (Weinstein, M. C. Ed. Clinical
Decision Analysis. Philadelphia: Saunders, pp. 84-108, 1980).
Stepwise logistic regression was used to establish the independent
predictors of cancer and to estimate the probability for
combinations of the MMP markers in the final multivariate model.
(Breslow, N. E. Statistical methods in cancer research. Volume 1.
Lyon, France: Int. Agency for Res. on Cancer, pp. 192 -210, 1980).
One-way analysis of variance was performed to assess differences in
creatinine levels among the groups with a Bonferroni correction for
multiple comparisons. Fisher's exact test was used for comparison
of proportions. All statistical tests were conducted at a two-sided
alpha level of 0.05. Data analysis, was performed using the SAS for
Windows statistical package version 6.11 (SAS Institute Inc., Cary,
N.C.).
[0095] Results shown in Tables 14 and 15 are consistent with
previous data presented herein with smaller samples. Specificity of
72 kDa MMP, for example, in prostatic cancer, all organ-confined
cancer and all cancers is found to be 100 at the 95% confidence
interval. For all cancers, the positive likelihood ratios (ratio of
true positives to false positives) of the high molecular weight
(.gtoreq.150 kDA) and 92 kDa MMPs are 8.4 and 6.3, respectively.
These results confirm the value of urine MMP zymogram routine
analysis to detect the presence of cancer in particular, as an
example of a tissue remodelling-associated condition, and for
monitoring of cancer patients during therapy, and for prognosis of
the course of cancer and the appearance of metastases.
Example 10
A Gelatinase Marker for Metastatic Breast Cancer
[0096] A gelatinase of approximately 125 kDa was detected in the
urine of 5 out of 9 specimens obtained from metastatic breast
cancer patients. Gelatinase of this size was not observed in urine
samples of other subjects.
Equivalents
[0097] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *