U.S. patent application number 13/198519 was filed with the patent office on 2011-12-01 for tissue inhibitor of matrix metalloproteinases type-1 (timp-1) as a cancer marker and postoperative marker for minimal residual disease or recurrent disease in patients with a prior history of cancer.
This patent application is currently assigned to HVIDOVRE HOSPITAL. Invention is credited to Nils Brunner, Ib Jarle Christensen, Mads Nikolaj Holten-Andersen, Hans Jorgen Nielsen.
Application Number | 20110294148 13/198519 |
Document ID | / |
Family ID | 8093987 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110294148 |
Kind Code |
A1 |
Holten-Andersen; Mads Nikolaj ;
et al. |
December 1, 2011 |
TISSUE INHIBITOR OF MATRIX METALLOPROTEINASES TYPE-1 (TIMP-1) AS A
CANCER MARKER AND POSTOPERATIVE MARKER FOR MINIMAL RESIDUAL DISEASE
OR RECURRENT DISEASE IN PATIENTS WITH A PRIOR HISTORY OF CANCER
Abstract
The present invention describes a method for determining whether
an individual is suffering from cancer by determining a parameter
representing the TIMP-1 concentration in body fluid samples from
the individual. The present invention furthermore describes a
method for determining whether an individual is suffering from
minimal residual disease or recurrent cancer after being treated
for the primary cancer by determining a parameter representing the
post-operative TIMP-1 concentration in body fluid samples from the
individual. In addition, the invention describes the additive
effect of combined post-operative measurements of plasma TIMP-1 and
serum CEA.
Inventors: |
Holten-Andersen; Mads Nikolaj;
(Vanlose, DK) ; Christensen; Ib Jarle; (Hillerod,
DK) ; Brunner; Nils; (Hellerup, DK) ; Nielsen;
Hans Jorgen; (Lyngby, DK) |
Assignee: |
HVIDOVRE HOSPITAL
Hvidovre
DK
RIGSHOSPITALET
Copenhagen
DK
|
Family ID: |
8093987 |
Appl. No.: |
13/198519 |
Filed: |
August 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12101661 |
Apr 11, 2008 |
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13198519 |
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10117030 |
Apr 8, 2002 |
7374886 |
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12101661 |
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09546573 |
Apr 10, 2000 |
7108983 |
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10117030 |
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Current U.S.
Class: |
435/7.94 ;
204/456; 702/179 |
Current CPC
Class: |
G01N 2333/96494
20130101; G01N 33/57415 20130101; G01N 33/57484 20130101; G01N
2333/8146 20130101; G01N 33/57419 20130101 |
Class at
Publication: |
435/7.94 ;
204/456; 702/179 |
International
Class: |
G01N 33/566 20060101
G01N033/566; G01N 27/447 20060101 G01N027/447; G06F 19/00 20110101
G06F019/00; G01N 33/559 20060101 G01N033/559 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 1999 |
DK |
199900476 |
Claims
1. A method for determining whether an individual is likely to have
minimal residual disease, recurrent cancer or a combination of
minimal residual disease and recurrent cancer, after being treated
for the primary cancer, the method comprising determining at least
one first parameter representing a post-operative concentration of
TIMP-1 in body fluid samples, and indicating the individual as
having a high likelihood of having minimal residual disease,
recurrent cancer or a combination of minimal residual disease and
recurrent cancer if the at least one first parameter is at or
beyond a discriminating value and indicating the individual as
unlikely of having minimal residual disease, recurrent cancer or a
combination of minimal residual disease and recurrent cancer if the
parameter is not at or beyond the discriminating value.
2. A method according to claim 1, wherein the discriminating value
is a value which has been determined by measuring said at least one
first parameter in both a healthy control population and in a
population with known minimal residual disease, recurrent cancer or
a combination of minimal residual disease and recurrent cancer,
thereby determining the discriminating value which identifies the
minimal residual disease, recurrent cancer or a combination of
minimal residual disease and recurrent cancer population with a
predetermined specificity or a predetermined sensitivity.
3. A method according to claim 1, wherein the discriminating value
is a value which has been determined by measuring said at least one
first parameter in a healthy control population, thereby
determining the discriminating value which identifies individuals
with increased plasma TIMP-1 values.
4. A method for determining whether an individual is likely to have
minimal residual disease, recurrent cancer or a combination of
minimal residual disease and recurrent cancer after being treated
for the primary cancer, the method comprising determining a
postoperative level of at least one first parameter representing
the concentration of TIMP-1 in body fluid samples, and indicating
the individual as unlikely of having minimal residual disease,
recurrent cancer or a combination of minimal residual disease and
recurrent cancer if the first parameter is below a discriminating
value, and indicating the individual as having a high likelihood of
having minimal residual disease, recurrent cancer or a combination
of minimal residual disease and recurrent cancer if the first
parameter is not below the discriminating value.
5. A method according to claim 4, wherein the discriminating value
is the preoperative level of said at least one first parameter
representing the concentration of TIMP-1 in body fluid samples.
6. A method according to claim 4, wherein the post-operative level
of the at least one first parameter representing the concentration
of TIMP-1 in body fluid samples is below the discriminating value
if the post-operative level of said first parameter is more than at
least 0.001% below the discriminating value.
7. A method according to claim 6, wherein the post-operative level
of said at least one first parameter is obtained 2 weeks after the
operation.
8. A method according to claim 1, wherein the at least one first
parameter is the total concentration of TIMP-1.
9. A method according to claim 1, wherein the at least one first
parameter is the value obtained by combining the concentration of
total TIMP-1 with the concentration of free TIMP-1.
10. A method according to claim 1, wherein the at least one first
parameter is the value obtained by combining the concentration of
total TIMP-1 with the concentration of a complex between TIMP-1 and
a specific MMP.
11. A method according to claim 1, wherein the at least one first
parameter is the value obtained by combining the concentration of
free TIMP-1 with the concentration of a complex between TIMP-1 and
a specific MMP.
12. A method according to claim 9, wherein the combining is
performed by logistic regression analysis.
13. A method according to claim 1, which comprises additionally
determining at least one second parameter, the second parameter
representing the concentration of an additional marker different
from any form of TIMP-1, in a body fluid sample from the
individual.
14. A method according to claim 13, wherein the at least one first
parameter representing the concentration of TIMP-1 in body fluid
samples and the at least one second parameter different from any
form of TIMP-1 are combined to result in a combined parameter and
indicating the individual as having a high likelihood of having
minimal residual disease, recurrent cancer or a combination of
minimal residual disease and recurrent cancer if the combined
parameter is at or beyond a discriminating value and indicating the
individual as unlikely of having minimal residual disease,
recurrent cancer or a combination of minimal residual disease and
recurrent cancer if the combined parameter is not at or beyond the
discriminating value.
15. A method according to claim 14, wherein the combining is
performed by logistic regression analysis.
16. A method according to claim 14, wherein the discriminating
value of the combined parameter is a value which has been
determined by determining said combined parameter in both a healthy
control population and a population with known minimal residual
disease, recurrent cancer or a combination of minimal residual
disease and recurrent cancer, thereby determining the
discriminating value which identifies the population with minimal
residual disease, recurrent cancer or a combination of minimal
residual disease and recurrent cancer with a predetermined
specificity or a predetermined sensitivity.
17. A method according to claim 14, wherein the discriminating
value of the combined parameter is a value which has been
determined by measuring said combined parameter in a healthy
control population, thereby determining the discriminating value
which identifies individuals with increased plasma TIMP-1
values.
18. A method according claim 13, wherein the at least one first
parameter representing the concentration of TIMP-1 in body fluid
samples and the at least one second parameter different from any
form of TIMP-1 are combined to result in a combined parameter and
indicating the individual as unlikely of having minimal residual
disease, recurrent cancer or a combination of minimal residual
disease and recurrent cancer if a post-operative value of the
combined parameter is below a discriminating value and indicating
the individual as having a high likelihood of having minimal
residual disease, recurrent cancer or a combination of minimal
residual disease and recurrent cancer if the post-operative value
of the combined parameter is not below the discriminating
value.
19. A method according to claim 18, wherein the discriminating
value is a pre-operative level of said combined parameter.
20. A method according to claim 18, wherein the post-operative
level of the combined parameter is below the discriminating value
if the post-operative level of the combined parameter is more than
at least 0.001% below the discriminating value.
21. A method according to claim 20, wherein the post-operative
level of the combined parameter is obtained 2 weeks after the
operation.
22. A method according to claim 18, wherein the combining is
performed by logistic regression analysis.
23. A method according to claim 13, wherein the at least one second
parameter determined is a parameter representing the concentration
of a tumour marker.
24. A method according to claim 23, wherein the tumour marker is
selected from the group consisting of CEA, soluble u-PAR, cathepsin
B, cathepsin H, HER2-neu, CA15-3 and YKL-40, 19.9 and CA242.
25. A method according to claim 24, wherein the at least one second
parameter determined is the concentration of CEA.
26. A method according to claim 1, wherein the individual is a
member of a population already identified as having an increased
risk of developing recurrent cancer.
27. A method according to claim 1, wherein the determination is
performed at several time points at intervals as part of a
monitoring of a cancer patient after the treatment for primary
cancer.
28. A method according to claim 1, wherein the body fluid is
selected from the group consisting of blood, serum, plasma, saliva,
faeces, urine and cerebrospinal fluid.
29. A method according to claim 28, wherein the body fluid is
plasma.
30. A method according to claim 28, wherein the body fluid is
blood.
31. A method according to claim 28, wherein the body fluid is
saliva.
32. A method according to claim 28, wherein the body fluid is
urine.
33. A method according to claim 1, wherein the concentration
determination is performed by means of an immuno assay or an
activity assay.
34. A method according to claim 33, wherein the immuno assay is an
ELISA.
35. A method according to claim 33, wherein the activity assay is
zymography.
36. A method according to claim 1, wherein the cancer is a
gastrointestinal cancer.
37. A method according to claim 36, wherein the cancer is selected
from the group consisting of colon cancer and rectal cancer.
38. A method according to claim 3, wherein the individuals with
increased TIMP-1 values are those whose values of the first
parameter are equal to or greater than the value of the first
parameter in 95 percentile of a healthy control population.
39. A method according to claim 7, wherein the post-operative value
of said first parameter is obtained at a time comprising 1 month
post-operation, 1.5 month postoperation, 2 month post-operation, 3
month post-operation, 4 month post-operation, 5 month
post-operation, 6 month post-operation, 7 month post-operation or 8
month post-operation.
40. A method according to claim 10, wherein the combining is
performed by logistic regression analysis.
41. A method according to claim 11, wherein the combining is
performed by logistic regression analysis.
42. A method according to claim 17, wherein the individuals with
increased plasma TIMP-1 values are those whose values of the
combined parameter are equal to or greater than the value of the
combined parameter in 95 percentile of a healthy control
population.
43. A method according to claim 21, wherein the post-operative
level of the combined parameter is obtained at a time comprising 1
month post-operation, 1.5 month postoperation, 2 month
post-operation, 3 month post-operation, 4 month post-operation, 5
month post-operation, 6 month post-operation, 7 month
post-operation or 8 month post-operation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 12/101,661, filed Apr. 11, 2008, which
is a divisional application of U.S. patent application Ser. No.
10/117,030 filed Apr. 8, 2002, now U.S. Pat. No. 7,374,886, which
was a continuation-in-part of U.S. application Ser. No. 09/546,573,
filed on Apr. 10, 2000, now U.S. Pat. No. 7,108,983. The entire
contents of U.S. application Ser. No. 10/117,030 are incorporated
herein by reference, and the entire contents of U.S. application
Ser. No. 09/546,573 are incorporated herein by reference to the
extent they are consistent with U.S. application Ser. No.
10/117,030 and with this application and invention. U.S.
application Ser. No. 09/546,573 claimed priority from Denmark
Application DK 1999 00476 filed Apr. 9, 1999.
BRIEF DESCRIPTION OF THE INVENTION
[0002] The present invention relates to a test to be used to screen
large populations for the occurrence of cancer. Furthermore it
relates to a method for monitoring colorectal cancer patients for
the persistency of minimal residual disease (MRD) or for the
recurrence of their cancer disease. The method is based on the
measurement of tissue inhibitor of metalloproteinases 1 (TIMP-1) in
body fluids either alone or in combination with other tumor
markers, e.g. Carcino Embryonic Antigen (CEA). The invention
permits the early identification of patients having colorectal
cancer and furthermore permits the identification of patients who
have persistent MRD or which patients will experience recurrence of
colorectal cancer, either as local recurrence or as distant
metastases. The method for identification of patients having
primary colorectal cancer is highly specific, and patients with
non-malignant conditions, such as inflammatory bowel diseases, are
not detected. Measurement of another similar inhibitor, TIMP-2,
does not demonstrate equivalent clinical value, indicating an
additional level of specificity of the invention.
[0003] The test is based on the measurement of tissue inhibitor of
metalloproteinases type 1 (TIMP-1), in various body fluids,
including blood, plasma, serum, saliva, stool, cerebrospinal fluid
and urine. TIMP-1 concentrations can be determined either as the
total TIMP-1 concentration, the free TIMP-1 concentration, the
concentration of complexes between TIMP-1 and Matrix
Metallo-Proteinases (MMPs) and/or ratios and fractions thereof,
hereafter referred to as TIMP-1 levels. According to the invention,
individuals with a high likelihood of having cancer, e.g.
colorectal cancer can be identified by elevated TIMP-1 levels in
their body fluids, while individuals with low TIMP-1 levels are
unlikely to suffer from cancer, e.g. colon cancer. Also according
to the invention, individuals with a prior diagnosis of cancer and
who have a high likelihood of later developing clinically manifest
recurrent cancer, e.g. gastrointestinal cancer such as colon
cancer, can be identified by elevated postoperative TIMP-1 levels
in their body fluids, while individuals with low postoperative
TIMP-1 levels are unlikely to suffer from or later develop
recurrent cancer. Thus, the invention can be used to identify
individuals with a high probability of having early stage,
non-symptomatic cancer, e.g. colon cancer or later developing
recurrent colorectal cancer. The identified individuals should be
further examined and if cancer, recurrent cancer or minimal
residual disease (MRD) or both recurrent cancer and MRD are found,
the patients should be offered surgery, irradiation,
anti-neoplastic therapy or any combination thereof, thereby
increasing the chance of cure and/or long term survival of the
individual.
BACKGROUND
[0004] Colorectal cancer is the fourth most frequent cancer in the
Western world, with about 160,000 new cases yearly in the US. Forty
to 50% of all colorectal cancer patients will be diagnosed with
early stage disease (Dukes' stage A or B). Most of these patients
with early stage colorectal cancer can be cured by surgery alone.
Thus, risk of recurrence is closely related to stage of disease at
time of primary surgery, with about a 10% relapse rate in Dukes'
stage A and 25-30% in Dukes' stage B. Patients with Dukes' stage C
colorectal cancer have a five-year relapse rate of 70% following
surgery and are offered adjuvant chemotherapy. Following relapse,
the risk of dying of the disease is significant. Thus, one way to
improve survival is to increase the number of patients being
diagnosed with early stage disease. Screening for colorectal cancer
has been shown to improve survival, however, current tests suffer
from a lack of compliance, from low sensitivity, and from the need
for strict dietary restrictions. Thus, the development of new and
improved tests for the early detection of colorectal cancer is
needed.
[0005] Thus, 30-40 percent of patients who have undergone a
complete resection of a colorectal malignancy will experience
relapse with metastatic disease, which is usually fatal. Thus,
hundreds of thousands of people with resected CRC are candidates
for surveillance, as it is a commonly held clinical view that early
detection of metastases and metachronous disease by relevant
surveillance regimens may allow for interventions with the aim of
improving overall survival, disease-free survival and quality of
life.
[0006] The majority of recurrences of CRC occur within 5 years, and
usually within 3 years following surgery. A tremendous effort has
been put into the establishment of effective tests for the
detection of recurrent disease at a stage when intervention is not
futile. Carcinoembryonic antigen (CEA) tests, colonoscopies, chest
x-rays, liver function tests, complete blood cell counts, fecal
occult blood tests, computerized tomography (CT), ultrasonography,
magnetic resonance (MR) and positron emission tomography (PET) are
all methods that have been extensively reported on for the
postoperative surveillance of CRC. Unfortunately, the results from
randomised studies of such monitoring of CRC patients have varied
widely and demonstrated minimal efficacy (Schoemaker, Gastroent,
1998; Ohlsson, Dis Col Rec, 1995; Makela, Arch Surg, 1996;
Kjeldsen, Int J Color Dis, 1997). As a result hereof, considerable
variation in clinical follow-up practice is evident (Virgo K S, Ann
Surg, 1995) and expenses for 5-year follow-up of CRC patients have
differed from $561 to $16,492 per patient (Virgo, JAMA, 1995).
[0007] The American Society of Clinical Oncology (ASCO) has
recommended against the use of liver function tests, fecal occult
blood tests, complete blood counts, pelvic imaging, CT scanning and
chest x-rays as means for regular postoperative monitoring of CRC.
However, it was recommended that postoperative measurement of serum
CEA is performed every 2 to 3 months for .gtoreq.2 years in
patients with stage II or III disease where resection of liver
metastases is clinically indicated (Benson, JCO, 2000). Of note,
however, is the fact that approximately 30 percent of all CRC
recurrences do not produce CEA (Safi, Cancer Detect Prev, 1993).
Thus, it was stressed by the ASCO that new surveillance methods for
the detection of CRC recurrences are needed (Benson, JCO, 2000).
This statement has gained further weight as recent data has shown
that chemotherapy of metastatic CRC can improve short-term survival
and often improve the quality of life (Cunningham, Lancet, 1998)
and that treatment of asymptomatic CRC yields better results than
postponing treatment until the disease has become symptomatic
(Nordic Gastr Tumor Adj Group, JCO, 1992).
[0008] Because metastatic disease is the main cause of cancer
patient morbidity and mortality, molecules involved in the
regulation of tumor invasion and metastasis are attractive as
potential diagnostic/prognostic/monitoring targets. It is well
established that proteolytic enzymes produced by cancer cells or by
cells in the tumor stroma are involved in extracellular tissue
degradation, leading to cancer cell invasion and metastasis. A
number of enzymes have been associated with this process, the most
thoroughly investigated being the metalloproteinases, such as the
collagenases and stromelysins, and the serine proteases such as
plasmin. Recently, data have been published indicating that these
molecules, free or bound in complexes, are released from tumor
tissue and find their way into the circulation.
[0009] Matrix metalloproteinases (MMP's) play a pivotal role in
cancer growth and spread, contributing to enzymatic degradation of
the extracellular matrix (Liotta et. al., 1991; Stetler-Stevenson
et. al., 1993; MacDougall & Matrisian, 1995). The naturally
occurring inhibitors of MMP's, tissue inhibitors of MMP's (TIMP's),
form tight 1:1 stoichiometric complexes with the activated forms of
the MMP's (Welgus et. al., 1985; Kleiner et. al., 1993), thereby
inhibiting the catalytic activity of these enzymes
(Stetler-Stevenson et. al., 1996; Goldberg et. al., 1992;
Birkedal-Hansen et. al., 1993). While the balance between the
matrix-degrading properties of MMP's and the inhibitory effect of
TIMP's is closely regulated under normal physiological conditions
(Matrisian, 1992; Thorgeirsson et. al., 1993; Birkedal-Hansen et.
al., 1993), this balance might be disrupted in malignant
tissue.
[0010] A number of enzyme-linked immunoassays for the detection of
TIMP-1 (Kodama et. al., 1989; Cooksley et. al., 1990; Clark et.
al., 1991) and TIMP-2 (Fujimoto et. al., 1993) have been described.
These assays have been applied to body fluids, e.g. serum, plasma,
amniotic fluid, cerebrospinal fluid, urine, but the number of
samples tested has not been sufficient to establish normal ranges
for TIMP levels in healthy individuals (Kodama et. al., 1989; Clark
et. al., 1991). Furthermore, none of these assays have been
sufficiently validated for technical performance or for clinical
use. We have recently described and validated an ELISA for the
quantitation of total TIMP-1 in plasma samples, and using this
assay we could demonstrate that healthy blood donors have a very
narrow range of total plasma TIMP-1 (Holten-Andersen et. al., Br.
J. Cancer 1999). We have also recently developed and validated an
ELISA for the quantitation of uncomplexed TIMP-1 in plasma
(Holten-Andersen et. al., manuscript submitted)
[0011] In a study by Mimori et. al. (Mimori et. al., 1997) in which
tumor tissue levels of TIMP-1 mRNA were studied in patients with
gastric carcinoma, high tumor/normal tissue ratios of TIMP-1 mRNA
were found to be associated with increased invasion and poor
prognosis and Guillem et. al. (Proc Annu Meet Am Assoc Cancer Res;
34:A466, 1993) suggest a possible correlation between TIMP-1 RNA
levels and poorly differentiated, invasive colorectal cancers.
However, TIMP-1 protein levels in sera from prostate cancer
patients and healthy donors (Baker et. al., 1994) showed a high
degree of overlap. Similarly, a separate study of plasma from
prostate cancer patients and healthy donors showed no difference in
TIMP-1 levels between the two groups (Jung et. al., 1997).
[0012] We have recently published that preoperatively measured
plasma levels of total TIMP-1 show highly significant association
with colorectal cancer patient survival, i.e. patients with high
TIMP-1 levels had a significantly shorter survival than those
patients with low TIMP-1 levels (Holten-Andersen et. al., Clin.
Cancer Res, 2000).
[0013] Furthermore, Nauro et. al. (J. Urol, vol. 1, no 3, September
1994, pp 228-231) found that serum TIMP-1 levels are significantly
higher in metastatic bladder cancer patients compared to a healthy
control group, whereas no results were presented about the TIMP-1
levels of pre-metastatic bladder cancers. TIMP-1 measurements in
urine have also been described as being useful in the diagnosis of
other cancers such as bladder cancer, liver cancer and renal cancer
(Japanese patent application no. 8-136548). In none of these
studies a correlation between the TIMP-1 level of patients with
early stage bladder cancers and the TIMP-1 level of patients with
cystitis have been included.
[0014] Measurements of TIMP-1 levels have also been shown to be
useful in diagnosing diseases of the nervous system as described in
U.S. Pat. No. 5,324,634.
[0015] Studies of TIMP-1 complexed with MMP-9 in plasma of patients
with advanced gastrointestinal and gynaecological cancer (Zucker
et. al., 1995) demonstrated significantly higher levels in blood
samples from cancer patients with metastatic disease compared to
healthy control individuals, and that patients with high levels of
TIMP-1:MMP-9 complex had a shorter survival (Zucker et. al., 1995
and U.S. Pat. No. 5,324,634). However, this study did not include
measurements of total or free TIMP-1, only the complex between
TIMP-1 and one of the up to now approximately 24 identified MMP's.
Furthermore, in this study, no differences in complex levels were
found between patients with breast cancer and healthy donors. Also,
this study did not include patients with early stage cancer.
DETAILED DESCRIPTION OF THE INVENTION
[0016] In a number of cancer types, there is a critical and unmet
need for highly sensitive and specific markers for screening large
populations for the presence of malignant disease. Such markers are
able to identify individuals with a high probability of early stage
cancer. These individuals should be further examined, and if cancer
is found, they should subsequently be offered surgery, radiation,
or adjuvant anti-neoplastic therapy or any combination thereof,
e.g. both surgery and radiation, surgery and systemic
anti-neoplastic therapy (chemotherapy).
[0017] Furthermore, in a number of cancer types, there is also a
critical and unmet need for highly sensitive and specific markers
for early detection of MRD or recurrent cancer disease. Such
markers are able to identify individuals with a high risk of
recurrent cancer. If positive test results are obtained, these
individuals should be further examined, and if minimal residual
disease (MRD) or recurrent cancer or both MRD and recurrent cancer
is found, the patients should subsequently be offered surgery,
radiation, systemic anti-neoplastic therapy or any combination
hereof, e.g. both surgery and radiation, surgery and systemic
anti-neoplastic therapy (chemotherapy).
[0018] Since proteinases and their receptors and inhibitors seem to
play a pivotal role in the basic mechanisms leading to cancer
invasion, these molecules may be expressed at a very early time
point in the carcinogenic process such as the invasion and
metastasis process. As many of these molecules exert their
biological action extracellularly, they may be present at elevated
levels in body fluids, even in patients with early stage invasive
malignant disease. Moreover, since these molecules are involved in
the more basic features of malignant progression, e.g. invasion and
metastasis, it should be investigated which forms of cancer that
display an increase in these molecules and furthermore in patients
with minimal residual disease after the primary treatment.
[0019] The present invention relates to a method to aid in the
diagnosis of primary colorectal cancer and furthermore to aid in
the diagnosis of MRD or recurrent cancer or a situation of both MRD
and recurrent cancer in a patient, said method comprising
determining the amount of total, complexed and/or free TIMP-1
levels and ratios and fractions thereof in body fluids such as
blood, serum, plasma, saliva, urine, faeces or cerebrospinal fluid.
These measurements can be combined with measurements of CEA or
other markers of recurrent colorectal cancer and additive
diagnostic effect may thus be obtained.
[0020] Gastrointestinal cancers such as colon cancer, rectal
cancer, colorectal cancer, pancreatic cancer, stomach (Gastric)
cancer, esophageal cancer, liver cancer or bladder cancer are
preferred types of cancer wherein the present method can be
applied.
[0021] An aspect of the present invention relates to a method for
determining whether an individual is likely to have cancer, the
method comprising determining a first parameter representing the
concentration of TIMP-1 in body fluid samples, and indicating the
individual as having a high likelihood of having cancer if the
parameter is at or beyond a discriminating value and indicating the
individual as unlikely of having cancer if the parameter is not at
or beyond the discriminating value.
[0022] The first parameter may be obtained by combining the
concentration of total TIMP-1 with the concentration of free
TIMP-1. The combination is performed by logistic regression
analysis. In an aspect of the invention, at least one second
parameter is additionally determined, which represents the
concentration of an additional marker different from any form of
TIMP-1, in a body fluid sample from the individual. The first
parameter representing the concentration of TIMP-1 in a body fluid
sample of an individual and the at least one second parameter
(different from any form of TIMP-1 in the individual's body fluid
sample) may be combined to produce a combined parameter. If the
combined parameter is at or beyond a discriminating value, the
individual is indicated as having a high likelihood of having
cancer. If the combined parameter is not at or beyond the
discriminating value, the individual is unlikely to have cancer. In
this embodiment, the discriminating value of the combined parameter
is a value determined by determining the combined parameter in both
a healthy control population and a population with known cancer.
Thus, the discriminating value identifies the cancer population
with a predetermined specificity or a predetermined
sensitivity.
[0023] The at least one second parameter may be a parameter
representing the concentration of a tumour marker. Such tumour
marker may be selected from the group consisting of CEA, soluble
u-PAR, cathepsin B, HER2-neu, CA 15-3 and YKL-40.
[0024] The combining to generate any combined parameter may be
performed by logistic regression analysis.
[0025] The method of the invention can be applied to various
cancers, such as colorectal cancers and metastatic breast cancer
(i.e., breast cancer in patients who have been previously treated
for breast cancer). In one embodiment directed to screening an
individual for metastatic breast cancer, the determination of the
appropriate parameter is performed at several points in time at
intervals, as part of monitoring of a cancer patient after the
treatment for primary cancer.
[0026] A further aspect of the present invention relates to a
method for determining whether an individual is likely to have MRD
or recurrent gastrointestinal cancer, such as colorectal cancer,
the method comprising determining a first parameter representing
the concentration of TIMP-1 in body fluid samples, and indicating
the individual as having a high likelihood of having MRD or
recurrent cancer or both MRD and recurrent cancer if the parameter
is at or beyond a discriminating value and indicating the
individual as unlikely of having recurrent cancer or MRD if the
parameter is not at or beyond the discriminating value.
[0027] The parameter representing the concentration of TIMP-1 may
be the concentration proper of TIMP-1. TIMP-1 exists both in free
form and in the form of complexes with MMPs, and it has been found
that an important parameter is the total concentration of TIMP-1,
that is, the sum of the TIMP-1 in free form (also referred to
herein as "free TIMP-1") and the TIMP-1 in complex forms. The
"total concentration of TIMP-1" may also be referred to herein as
"concentration of total TIMP-1", or in similar expressions. It will
be understood that the other expressions than the concentration
proper can represent the concentration, such as, e.g., the
concentration multiplied by a factor, or similar expressions, and
that such other representations can be used equally well for the
purpose of the present invention provided the corresponding
adjustments are made.
[0028] The discriminating value is a value which has been
determined by measuring the parameter in both a healthy control
population and a population with known cancer thereby determining
the discriminating value which identifies the cancer population
with either a predetermined specificity or a predetermined
sensitivity based on an analysis of the relation between the
parameter values and the known clinical data of the healthy control
population and the cancer patient population, such as it is
apparent from the detailed discussion in the examples herein. The
discriminating value determined in this manner is valid for the
same experimental setup in future individual tests.
[0029] In the case of identifying individuals who are likely to
have MRD or recurrent gastrointestinal cancer, the discriminating
value is a value which has been determined by measuring the
parameter in both a healthy control population and a population
with known MRD or recurrent cancer thereby determining the
discriminating value which identifies the cancer population with
either a predetermined specificity or a predetermined sensitivity
based on an analysis of the relation between the parameter values
and the known clinical data of the healthy control population and
the cancer patient population, such as it is apparent from the
detailed discussion in the examples herein. The discriminating
value determined in this manner is valid for the same experimental
setup in future individual tests.
[0030] Preferred embodiments of the present invention are described
below.
[0031] The present invention relates in general to a method for
determining whether an individual is likely to have minimal
residual disease and/or recurrent cancer after being treated for
the primary cancer, said primary cancer being the possible
progenitor to the minimal residual disease or recurrent cancer, the
method comprising determining a first parameter representing the
post-operative concentration of TIMP-1 in body fluid samples, and
indicating the individual as having a high likelihood of having
minimal residual disease and/or recurrent cancer if the parameter
is at or beyond a discriminating value and indicating the
individual as unlikely of having minimal residual disease or
recurrent cancer if the parameter is not at or beyond the
discriminating value.
[0032] In a preferred embodiment the discriminating value is a
value which has been determined by measuring said at least one
first parameter in both a healthy control population and in a
population with known minimal residual disease and/or recurrent
cancer, thereby determining the discriminating value which
identifies the minimal residual disease and/or recurrent cancer
population with a predetermined specificity or a predetermined
sensitivity.
[0033] In an equally preferred embodiment the discriminating value
is a value which has been determined by measuring said at least one
first parameter in a healthy control population, thereby
determining the discriminating value which identifies individuals
with increased plasma TIMP-1 values (e.g., above or equal to the
95.sup.th percentile of healthy control individuals). The term
"95.sup.th percentile of healthy control individuals", as used
herein, indicates the TIMP-1 value which dichotomises the
individuals so that 5% thereof have higher TIMP-1 levels and 95%
lower TIMP-1 levels.
[0034] In an additional embodiment the present invention relates to
a method for determining whether an individual is likely to have
minimal residual disease and/or recurrent cancer after being
treated for the primary cancer, said primary cancer being the
possible progenitor to the minimal residual disease or recurrent
cancer, the method comprising determining the postoperative level
of a first parameter representing the concentration of TIMP-1 in
one or more body fluid samples, and indicating the individual as
unlikely of having minimal residual disease and/or recurrent cancer
if the first parameter is below a discriminating value where this
discriminating value is the pre-operative level of said first
parameter representing the concentration of TIMP-1 in one or more
body fluid samples and indicating the individual as having a high
likelihood of having minimal residual disease or recurrent cancer
if the first parameter is not below this discriminating value.
[0035] The phrase "below the discriminating value" is meant to
identify the situation where the postoperative level of said first
parameter is more than at least 0.001, at least 0.01, at least 0.1
or at least 1% below the pre-operative level.
[0036] Furthermore the post-operative level of said first parameter
may be obtained after the operation, such as at least 1 day
post-operatively, such as 5 days post-operatively, e.g. 1 week
post-operatively, e.g. 2 weeks post-operatively, e.g. 1 month
post-operatively, e.g. 1.5 month post-operatively, e.g. 2 months
post-operatively, e.g. 3 months post-operatively, e.g. 4 months
post-operatively, e.g. 5 months post-operatively, e.g. 6 months
post-operatively, e.g. 7 months post-operatively, such as 8 months
post-operatively.
[0037] It is obvious that the determination of TIMP-1 level with
advantage can be performed at several time points at intervals as
part of the monitoring of a cancer patient after the treatment for
primary cancer for as long as it is found relevant, and that this
recurrent determination of the TIMP-1 level can be performed
according to any one of the herein described embodiments.
[0038] In a preferred embodiment the first parameter determined is
the total concentration of TIMP-1.
[0039] In a further preferred embodiment the at least one first
parameter determined is the value obtained by combining the
concentration of total TIMP-1 with the concentration of free
TIMP-1, alternatively the at least one first parameter determined
may be the value obtained by combining the concentration of total
TIMP-1 with the concentration of a complex between TIMP-1 and a
specific MMP or the at least one first parameter determined may be
the value obtained by combining the concentration of free TIMP-1
with the concentration of a complex between TIMP-1 and a specific
MMP. The combined values are obtained by performing logistic
regression analysis.
[0040] A special embodiment of the present invention is the method
which comprises additionally determining at least one second
parameter, the second parameter representing the concentration of
an additional marker different from any form of TIMP-1, in a body
fluid sample from the individual.
[0041] This results in a method wherein the first parameter
representing the concentration of TIMP-1 in body fluid samples and
the at least one second parameter different from any form of TIMP-1
are combined to result in a combined parameter and indicating the
individual as having a high likelihood of having minimal residual
disease and/or recurrent cancer if the combined parameter is at or
beyond a discriminating value and indicating the individual as
unlikely of having minimal residual disease and/or recurrent cancer
if the combined parameter is not at or beyond the discriminating
value.
[0042] Again, the combined values are obtained by performing
logistic regression analysis.
[0043] In the embodiment where at least one second parameter is
combined with measurement of TIMP-1, the discriminating value of
the combined parameter may be a value which has been determined by
determining said combined parameter in both a healthy control
population and a population with known minimal residual disease
and/or recurrent cancer, thereby determining the discriminating
value which identifies the population with minimal residual disease
and/or recurrent cancer with a predetermined specificity or a
predetermined sensitivity.
[0044] Furthermore, the discriminating value of the combined
parameter may be a value which has been determined by measuring
said combined parameter in a healthy control population, thereby
determining the discriminating value which identifies individuals
with increased values of the combined parameter (e.g. above or
equal to the 95.sup.th percentile of healthy control individuals).
Thus, the individuals with increased combined parameter values are
those having combined parameter values equal to or greater than 95%
of the combined parameter values in the healthy control
individuals.
[0045] A further embodiment of the present invention is a method
wherein the first parameter representing the concentration of
TIMP-1 in body fluid samples and the at least one second parameter
different from any form of TIMP-1 are combined to result in a
combined parameter and indicating the individual as unlikely of
having minimal residual disease and/or recurrent cancer if the
post-operative level of the combined parameter is below the
pre-operative level of said combined parameter and indicating the
individual as having a high likelihood of having minimal residual
disease and/or recurrent cancer if the post-operative level of the
combined parameter is not below the pre-operative level of said
combined parameter.
[0046] The phrase "below the discriminating value" is meant to
identify the situation where the postoperative level of the
combined parameter is more than at least 0.001, at least 0.01, at
least 0.1 or at least 1% below the pre-operative level of said
combined parameter.
[0047] Again, the post-operative level of said combined parameter
may be obtained after the operation, such as at least 1 day
post-operatively, such as 5 days post-operatively, e.g. 1 week
post-operatively, e.g. 2 weeks post-operatively, e.g. 1 month
post-operatively, e.g. 1.5 month post-operatively, e.g. 2 months
post-operatively, e.g. 3 months post-operatively, e.g. 4 months
post-operatively, e.g. 5 months post-operatively, e.g. 6 months
post-operatively, e.g. 7 months post-operatively, such as 8 months
post-operatively.
[0048] In a preferred embodiment of the present invention, the at
least one second parameter determined is a parameter representing
the concentration of a tumour marker where the tumour marker is
selected from the group consisting of CEA, soluble u-PAR, cathepsin
B, cathepsin H, HER2-neu, CA15-3 and YKL-40, 19.9 and CA242. In a
more preferred embodiment, the at least one second parameter
determined is the concentration of CEA.
[0049] In general, the method of the present invention relates to
the situation where the individual may be a member of a population
already identified as having an increased risk of developing
recurrent cancer.
[0050] It is obvious that the determination of the TIMP-1 level or
the determination of the combination of TIMP-1 level and the level
of a non-TIMP-1 marker may be performed at several time points at
intervals as part of a monitoring of a cancer patient after the
treatment for primary cancer.
[0051] The determination of the concentration of the markers,
either TIMP-1 or any of the non-TIMP-1 markers may performed by
means of an immuno assay, such as ELISA, or an activity assay, such
as zymography.
[0052] "Sensitivity" is defined as the proportion of positives
(i.e. individuals having a parameter representing the concentration
of a marker, such as TIMP-1, in body fluid samples higher than a
predefined diagnostic level) that are correctly identified by the
described method of the invention as having MRD and/or recurrent
cancer. "Specificity" is defined as the proportion of negatives
(i.e. individuals having a parameter representing the concentration
of a marker, such as TIMP-1, in body fluid samples lower than a
predefined diagnostic level) that are correctly identified by the
described method as not having the disease.
[0053] The invention may be used both for an individual and for an
entire population.
[0054] In the specific experimental setups described herein, the
concentration threshold of total TIMP-1 useful as a discriminating
value was found to be 134 microgram/L of total TIMP-1 which is the
95.sup.th percentile of healthy donors. Other experimental setups
and other parameters will result in other values
(specificities/sensitivities) which can be determined in accordance
with the teachings herein.
[0055] An important point is that the present invention is not
directed to such a single, precise value of TIMP-1 concentration
representing the discriminating value. A relevant discriminating
value of the TIMP-1 concentration can be selected from ROC
(Receiver Operating Characteristics) curves which are disclosed
e.g. in FIG. 13. These curves give the correlation between
sensitivity and specificity and the sensitivity/specificity for any
selected total TIMP-1 threshold value can be derived from the
curves. A threshold value resulting in a high sensitivity results
in a lower specificity and vice versa. For example, if one wishes
to detect all colorectal cancers with a high degree of certainty,
then the specificity will be lower and some false positives are
likely to be included. If one wishes to be relatively certain that
only malignant colorectal cancers will be detected, then a number
of non-malignant colorectal cancers are likely not to be
identified.
[0056] Each individual diagnostic department or a person thus can
determine which level of sensitivity/specificity is desirable and
how much loss in specificity is tolerable. The chosen
discriminating value could be dependent on other diagnostic
parameters used in combination with our claimed method by the
individual diagnostic department, e.g. the use of colonoscopy.
[0057] The method can be applied to an unselected population, but
more appropriately to a population already identified as having an
increased risk of developing cancer, e.g. individuals with a
genetic disposition, individuals who have been exposed to
carcinogenic substances, or individuals with cancer-predisposing,
non-malignant diseases. In the case of colorectal cancer, the
population selected for the invention could represent individuals
with a prior polyp, individuals with Crohn's disease or ulcerative
colitis, individuals with one or more family members with
colorectal cancer, or individuals with a prior resection of an
early colorectal cancer.
[0058] When an individual has been identified as having high TIMP-1
levels in his or her body fluid, the individual should be referred
for further examination. If a cancer is found, the patient could be
offered surgery, radiation or adjuvant anti-neoplastic therapy
aiming at curing the patient of cancer.
[0059] In the case of identifying individuals who are likely to
have MRD or recurrent gastrointestinal cancer, the method can be
applied to all patients who have had primary treatment for
colorectal cancer.
[0060] When an individual who is likely to have MRD or recurrent
gastrointestinal cancer has been identified as having high TIMP-1
levels in his or her body fluid, the individual should be referred
for further examination. If MRD and/or a recurrent cancer is found,
the patient could be offered surgery, radiation and/or systemic
anti-neoplastic therapy or any combination thereof aiming at curing
the patient of cancer.
[0061] Example 1 describes the preparation and validation of an
assay that measures total TIMP-1 with high analytical sensitivity
and specificity. It is described that healthy blood donors have a
very narrow range of plasma total TIMP-1.
[0062] In Example 2, the formatting of a TIMP-1:MMP-9 ELISA is
described. The format, execution, and validation of this assay are
similar to those for total TIMP-1, except that a polyclonal
antibody against MMP-9 is used in the capture step. By substituting
the MMP-9 antibody with an antibody against another MMP, complexes
between TIMP-1 and other MMP's can be quantitated.
[0063] In Example 3, the formatting of a TIMP-1 assay which
exclusively measures free TIMP-1, is described. This assay utilizes
a monoclonal anti-TIMP-1 antibody (MAC 19) which only recognises
TIMP-1 in its uncomplexed form. Thus, this assay will measure the
amount of free TIMP-1 in a sample. The execution and validation of
this assay are similar to the assay for total TIMP-1.
[0064] By subtracting the free TIMP-1 concentration from the total
TIMP-1 concentration in a biological sample, the concentration of
all complexed forms of TIMP-1 can be determined. It should be
emphasized, however, that TIMP-1 can form complexes with many of
the MMP's and therefore, subtracting one type of complex from the
total amount of TIMP-1 will only provide information on the
fraction of TIMP-1 not being complexed to this specific MMP.
[0065] In Example 4, which includes data regarding plasma TIMP-1
levels from healthy blood donors and from patients with known
colorectal cancer it is shown that patients suffering from
colorectal cancer have significantly elevated total TIMP-1 levels
in their preoperative plasma samples. A percentile plot of the
total TIMP-1 levels in plasma from all colorectal cancer patients
and from healthy blood donors shows that a total TIMP-1
concentration of 119.1 .mu.g/L is the 90.sup.th centile of the
healthy donors. Using this cut-off, 68% of the colorectal cancer
patients were identified as having elevated plasma TIMP-1 levels.
When analyzing the colon cancer patients separately, it was shown
that total TIMP-1 measurements in plasma identified 75% of the
colon cancer patients (sensitivity) with a 90% specificity (10% of
the healthy blood donors were classified as being high). Similarly,
it was shown for the rectal cancer patients alone, that total
TIMP-1 measurements in plasma identified 60% of the rectal cancer
patients with a 90% specificity. If a higher or lower sensitivity
or specificity is desired, the cut-off value can be changed. This
is illustrated in FIG. 13 showing ROC curves of total TIMP-1 in
plasma from colorectal cancer patients. In addition, ROC curves are
included for the individual groups of colon and rectal cancer
patients. Any other information which can be derived from these ROC
curves falls within the scope of the present invention.
[0066] An independent prospective study, including preoperative
plasma samples from 64 patients with colon (n=43) or rectal (n=21)
cancer confirmed the data obtained from the above described study
(FIG. 15). An additional study of 180 healthy blood donors and 20
colorectal cancer patients, using different antibodies (Anti TIMP-1
11E/C6, Anti-TIMP-1 RRU-T6) in an automated immunoassay, further
corroborated the previous clinical results. Moreover, the absolute
values generated from the automated assay showed a high degree of
correlation to those obtained by the assay described in Example 1
(r=0.9).
[0067] The clinical value of a marker for cancer detection or
screening is related to its ability to detect early stages of
disease, potentially impacting survival. It was shown that total
TIMP-1 was as efficient in detecting or screening early stage
colorectal cancer (Dukes' stages A and B) as it was in the total
population of colorectal cancer patients (FIG. 14). Thus, detection
or screening with total TIMP-1 will result in more patients being
diagnosed with early stage cancer. In a similar manner, any
information that can be derived from FIG. 14 falls within the scope
of the present invention.
[0068] By dividing the colon cancer patients into two groups,
patients with left-sided and right-sided tumors respectively, it
was evident that measurement of total TIMP-1 was especially useful
in identifying those patients with early stage, right-sided colon
cancer lesions (FIG. 14).
[0069] The specificity of a given cancer detection or screening
test is based on the efficiency of the test to identify only those
patients suffering from cancer while patients suffering from
non-malignant diseases should not be identified as false positive
subjects. In the case of colorectal cancer, it is important that
the test in question can distinguish between malignant and
non-malignant diseases of the colon and rectal. This is
particularly important for diseases like Crohn's disease and
ulcerative colitis, since patients with these diseases are at
higher risk of developing cancer.
[0070] In Example 5 it is shown that total TIMP-1 levels are
significantly higher in patients with colorectal cancer than in
patients with inflammatory bowel diseases (IBD), showing that total
TIMP-1 can be used to detect or screen for colorectal cancer in a
population of patients with IBD. That TIMP-1 is not increased in
non-malignant diseases is supported by a recent paper, (Keyser et.
al., 1999), demonstrating that patients with rheumatoid arthritis
do not have increased plasma TIMP-1 levels. Also, by comparing
total TIMP-1 levels among patients with IBD (excluding patients
with clinically assessed acute active disease, n=4) and healthy
blood donors, no significant differences in total plasma TIMP-1
levels were found (p=0.56), showing that these non-malignant
diseases do not give false positive test results.
[0071] In Example 6, the additive effect of the measurement of an
additional colorectal cancer marker is described. Carcino Embryonic
Antigen (CEA) was measured in all cancer patients and healthy blood
donor samples. Combining CEA and the TIMP-1 levels measured by the
assay described in Example 1, it could be shown that while CEA
alone gives 35% sensitivity at a 98% specificity, the sensitivity
of the combination of CEA and total TIMP-1 as determined by
logistic regression analysis increased to 57%, without sacrificing
specificity.
[0072] In Example 7 it is shown that patients suffering from
colorectal cancer have significantly elevated free TIMP-1 levels in
their preoperative plasma samples. A percentile plot including the
free TIMP-1 levels from the colorectal cancer patients as well as
free TIMP-1 levels from healthy blood donors, showed that
colorectal cancer patients had significantly elevated free TIMP-1
levels as compared to blood donors p=0.02. A ROC curve was created
(FIG. 18) for these patients and donors and it was seen that free
TIMP-1 gave an area under the curve (AUC) of 0.61.
[0073] In Example 8, the use of the TIMP-1:MMP-9 complex assay as
an aid for the detection or screening of colorectal cancer is
described.
[0074] TIMP-1 is known to exist either as the free molecule or in
complex with MMP's, preferentially MMP-9. Measuring total TIMP-1,
complexed TIMP-1 and free TIMP-1 will make it possible to validate
each of these species for their potential detection or screening
value. In addition, it will be possible to calculate ratios or any
derived algorithm between the different species which might provide
additional detection or screening value.
[0075] In Example 9, the detection or screening value of the
combination of total and free TIMP-1 is described.
[0076] The data shows that combining by logistic regression
analysis the free TIMP-1 measurements with the total TIMP-1
measurements, a significant increase in the AUC was demonstrated.
Any information that can be derived from FIG. 18 falls within the
scope of the present invention.
[0077] The specificity of a given cancer screening test is based on
the efficacy of the test to identify only those patients suffering
from cancer while patients suffering from non-malignant diseases
should not be identified as false positive. However, it would be
desirable that the test was specific for a specific type of cancer,
e.g. colon cancer, instead of being a pan-cancer marker.
[0078] In Example 10, total plasma TIMP-1 values in preoperative
blood samples from a cohort of 322 patients with primary breast
cancer (stage I and II) as compared with total TIMP-1 levels in 108
healthy blood donors are described. It was shown that the breast
patients had a median, total TIMP-1 level of 88.3 .mu.g/L, while
the healthy donors had a median plasma concentration of total
TIMP-1 of 88.9 .mu.g/L. The difference between these values is not
clinically significant, supporting the specificity of TIMP-1 levels
for the detection or screening of colorectal cancer. However, it
should be studied whether elevated plasma TIMP-1 levels are found
in patients with early stage non-colorectal cancer.
[0079] In Example 11, total TIMP-1 levels in patients with
metastatic breast cancer are described.
[0080] Of note, total TIMP-1 in plasma from women with metastatic
breast cancer had a median value of 236 .mu.g/L, significantly
higher than levels in healthy blood donors. This shows the
potential of using plasma TIMP-1 levels for the management of
breast cancer patients.
[0081] In Example 12, the concentrations of TIMP-2 in preoperative
plasma samples from patients with colorectal cancer are
described.
[0082] TIMP-2 is another tissue inhibitor of metalloproteinases
with a high degree of homology to TIMP-1. Using a specific
immunoassay for TIMP-2, concentrations of this inhibitor were
determined in plasma samples from colorectal cancer patients and in
healthy blood donors. No significant differences in plasma TIMP-2
levels were found between the two populations, supporting the
unique value of TIMP-1 as an aid for the early detection or
screening of colorectal cancer.
[0083] In Example 13, it is shown that a proportion of patients who
have previously had surgery for primary colorectal cancer may have
elevated plasma TIMP-1 levels 6 month post-surgery.
[0084] Example 14 shows that as an alternative to a fixed plasma
TIMP-1 threshold value, patients with a high likelihood of MRD or
recurrent cancer can also be identified as those patients who do
not show a significant decrease in plasma TIMP-1 levels between the
pre-operative plasma sample and the post-operative plasma
sample.
[0085] In Example 15, it is shown that the post-operative
information on recurrence probability obtained by measuring plasma
TIMP-1 6 months postoperatively, is independent of Dukes
classification, since statistically significant information on
survival can be obtained in each of Dukes stage A+B and C
disease.
[0086] In Example 16, it is shown that statistically significant
information on metastasis-free survival and overall survival can be
obtained by measuring plasma TIMP-1 and serum CEA 6 months
post-operatively. However, the information obtained by simultaneous
measuring plasma TIMP-1 and serum CEA is additive, e.g. significant
additive information is gained when combining these to serological
markers.
[0087] The clinical value of a marker for monitoring cancer
patients is related to its ability to detect recurrence of the
disease at an early time point when the patients still have no
symptoms or clinical signs of disease recurrence. From the
metastasis-free survival curves and the overall survival curves
presented, it can be deduced that plasma TIMP-1 measurements yields
highly statistically significant information on recurrence
probability several months before clinically evident recurrence is
observed. Thus, using TIMP-1, either alone or in combination with
CEA, will result in more patients being identified at an early time
point as having high probability of disease recurrence and these
patients might be offered systemic anti-cancer therapy. The benefit
to the patient is that treatment can be introduced at an early time
point where the tumor burden is still limited.
[0088] The specificity of a given cancer monitoring test is based
on the efficiency of the test to identify only those patients
suffering from recurrent cancer while patients suffering from
non-malignant diseases should not be identified as false positive
subjects. In the case of colorectal cancer, it is important that
the test in question can also distinguish between malignant and
non-malignant diseases of the colon and rectal. This is
particularly important for diseases like Crohn's disease and
ulcerative colitis, since patients with these diseases are at
higher risk of developing cancer.
[0089] In Example 17 it is shown that total TIMP-1 levels are
significantly higher in patients with recurrent colorectal cancer
than in patients with inflammatory bowel diseases (IBD), showing
that total TIMP-1 can be used to monitor for colorectal cancer
recurrence in a population of patients with IBD. That TIMP-1 is not
increased in non-malignant diseases is supported by a recent paper,
(Keyser et. al., 1999), demonstrating that patients with rheumatoid
arthritis do not have increased plasma TIMP-1 levels. Also, by
comparing total TIMP-1 levels among patients with IBD (excluding
patients with clinically assessed acute active disease, n=4) and
healthy blood donors, no significant differences in total plasma
TIMP-1 levels were found (p=0.56), showing that these non-malignant
diseases do not give false positive test results. In a recent paper
by Holten-Andersen et. al., 2002, it was shown that patients
(n=322) with primary breast cancer do not present with elevated
plasma-TIMP-1 levels.
[0090] TIMP-1 is known to exist either as the free molecule or in
complex with MMP's, preferentially MMP-9. Measuring total TIMP-1,
complexed TIMP-1 and free TIMP-1 will make it possible to validate
each of these species for their potential monitoring value. In
addition, it will be possible to calculate ratios or any derived
algorithm between the different species which might provide
additional monitoring value.
FIGURE LEGENDS
[0091] FIG. 1: Kinetic assay for TIMP-1. Progress curves for the
change in absorbance at 405 nm produced by hydrolysis of
p-nitrophenyl phosphate by solid-phase bound alkaline phosphatase
immunoconjugate. The data shown are generated by 4 individual assay
wells treated with 4 different concentrations of purified
recombinant TIMP-1; 10 .mu.g/L (.gradient.-.gradient.), 2.5 .mu.g/L
(.DELTA.-.DELTA.), 0.63 .mu.g/L (.quadrature.-.quadrature.) and
0.16 .mu.g/L (.largecircle.-.largecircle.). The lines shown have
been fitted by simple linear regression.
[0092] FIG. 2: TIMP-1 standard curve. Absorbance units for
triplicate TIMP-1 standards in the range of 0 to 5 .mu.g/L are
collected automatically over 60 minutes, with readings taken at 405
nm every 10 min. Progress curves are computed for each well and the
rates obtained are fitted to a standard curve using a
four-parameter equation of the form y=d+[(a-d)/(1+(x/c).sup.b)]. In
the example shown, the four derived parameters had the following
values: a=1.87, b=1.11, c=3.35, d=73.5. The correlation coefficient
for the fitted curve is >0.999.
[0093] FIG. 3: Recovery of signal from standard TIMP-1 added in
increasing concentration to assay dilution buffer
(.quadrature.-.quadrature.), a 1:100 dilution of EDTA plasma pool
(.DELTA.-.DELTA.), a 1:100 dilution of citrate plasma pool
(.gradient.-.gradient.) and a 1:100 dilution of heparin plasma pool
(.largecircle.-.largecircle.). The values shown are the means of
triplicates. The correlation coefficient for each fitted curve is
greater than 0.99.
[0094] FIG. 4: Western blotting of immunoabsorbed patient plasma
sample. Lane 1: standard TIMP-1; lane 2: eluate of patient citrate
plasma sample diluted 1:10 and immunoabsorbed with sheep polyclonal
anti-TIMP-1. Bands of non-reduced standard TIMP-1 and TIMP-1
isolated from plasma sample both appear just below 30 kDa.
[0095] FIG. 5a: Percentiles plot for the level of TIMP-1 (.mu.g/L)
measured in citrate plasma (.largecircle.) and EDTA plasma
(.DELTA.) from the same individual in a set of 100 volunteer blood
donors.
[0096] FIG. 5b: Linear regression plot for the level of TIMP-1 in
citrate plasma samples compared with EDTA plasma samples from the
same 100 individuals. The equation of the fitted line is y=0.93x,
with a regression coefficient of 0.99.
[0097] FIG. 6: Percentiles plot for the levels of TIMP-1 (.mu.g/L)
measured in two sets of citrate plasma samples obtained by the same
procedure from volunteer blood donors at different times. 100
samples from May=97 (.DELTA.) and 94 samples from September=96
(.quadrature.).
[0098] FIG. 7: Percentile plot for the levels of TIMP-1 (.mu.g/L)
measured in 194 citrate plasma samples from volunteer blood donors
and stratified by sex into 107 males (.DELTA.) and 87 females
(.largecircle.).
[0099] FIG. 8: Graphical illustration of the TIMP-1:MMP-9 complex
ELISA.
[0100] FIG. 9: Graphical illustration of the free TIMP-1 ELISA.
[0101] FIG. 10: A plate was coated with the polyclonal anti-MMP-9
antibody. Different concentrations of TIMP-1:MMP-9 complex, free
MMP-9, free TIMP-1 and a blank control were added. Only
TIMP-1:MMP-9 complexes and free MMP-9 were bound by the capture
polyclonal anti-MMP-9 antibody. MAC19 was then added for antigen
detection. Neither TIMP-1:MMP-9 complex nor free MMP-9 were
detected by MAC19, defining the specificity of this antibody for
free TIMP-1.
[0102] FIG. 11: A plate was coated with the polyclonal anti-MMP-9
antibody. Different concentrations of TIMP-1:MMP-9 complex, free
MMP-9, free TIMP-1 and a blank control were added. Only
TIMP-1:MMP-9 complexes and free MMP-9 were bound by the capture
polyclonal anti-MMP-9 antibody. MAC15 was then added for antigen
detection. Only TIMP-1:MMP-9 complex bound by the capture
polyclonal anti-MMP-9 antibody was detected by MAC15. Free MMP-9
was not detected by MAC15.
[0103] FIG. 12: Recovery of signal in the free TIMP-1 assay from
standard TIMP-1 added in increasing concentration to assay dilution
buffer (.quadrature.-.quadrature.), a dilution of EDTA plasma pool
(.DELTA.-.DELTA.), a dilution of citrate plasma pool
(.gradient.-.gradient.), and a dilution of heparin plasma pool
(.largecircle.-.largecircle.). The values shown are the means of
triplicates. The correlation coefficient for each fitted curve is
greater than 0.99.
[0104] FIG. 13: ROC curves for total TIMP-1 in all colorectal
cancer patients, in rectal cancer patients separately and in colon
cancer patients separately. As healthy control subjects, a cohort
of 108 healthy blood donors was used. (Number in parenthesis=Area
under curve)
[0105] FIG. 14: ROC curves for total TIMP-1 in all colorectal
cancer patients with Dukes' A or B disease. In addition, ROC curves
for Dukes' A or B patients with colon cancer or with right sided
colon cancer is included. (Number in parenthesis=Area under
curve)
[0106] FIG. 15: ROC curve for total TIMP-1 from an independent set
of 64 colorectal cancer patients compared to 108 healthy blood
donors is shown. (AUC=Area under curve)
[0107] FIG. 16: Box plot showing total TIMP-1 concentrations in
plasma from healthy blood donors, from patients with Crohn's
Disease, from patients with ulcerative colitis and from patients
with colorectal cancer. Medians, 10th, 25th, 75th and 90th centiles
are shown.
[0108] FIG. 17: ROC curves for total TIMP-1, CEA and for the
combination of total TIMP-1 and CEA in patients with right colon
cancer patients and 108 healthy blood donors. (Number in
parenthesis=Area under curve)
[0109] FIG. 18: ROC curves for plasma free TIMP-1, for plasma total
TIMP-1 and for the combination hereof in 64 CRC patients and 108
donors. (Number in parenthesis=Area under curve)
[0110] FIG. 19: ROC curve for 322 primary breast cancer patients
and 108 blood donors. (AUC=Area under curve)
[0111] FIG. 20: Percentiles plot for the level of total TIMP-1
(.mu.g/L) measured by ELISA in 19 EDTA plasma samples from female
breast cancer patients (.largecircle.) and 87 healthy blood donors
(.DELTA.).
[0112] FIG. 21: Percentile plot of total TIMP-1 in colorectal
cancer patients as measured 6 months postoperatively and in healthy
control subjects.
[0113] FIG. 22: Univariate survival analysis for total TIMP-1 in
colorectal cancer patients d. Preoperative and postoperative plasma
TIMP-1 levels were scored as low or high based on the 95th
percentile of plasma TIMP-1 in an age matched healthy control
group.
[0114] FIG. 23: Univariate survival curves. The patients were
dichotomised using the 95.sup.th percentile of plasma TIMP-1 levels
in healthy blood donors. Patient samples: 6 months postoperative
plasma TIMP-1 level. The Figure shows the survival curves for Dukes
A+B patients and for Dukes C patients, respectively.
[0115] FIG. 24: Univariate metastasis-free survival curves. The
patients were dicotomized into four groups according to their 6
months postoperative plasma TIMP-1 level and serum CEA level. Group
1: low TIMP-1 and low CEA; Group 2: high TIMP-1 and low CEA; Group
3: low TIMP-1 and high CEA; Group 4: high TIMP-1 and high CEA. The
patients were dichotomised using the 95.sup.th percentile of plasma
TIMP-1 levels in healthy blood donors and 5 ng/ml of CEA
[0116] FIG. 25: Univariate overall survival curves. The patients
were dicotomized into four groups according to their 6 months
postoperative plasma TIMP-1 level and serum CEA level. Group 1: low
TIMP-1 and low CEA; Group 2: high TIMP-1 and low CEA; Group 3: low
TIMP-1 and high CEA; Group 4: high TIMP-1 and high CEA. The
patients were dichotomised using the 95.sup.th percentile of plasma
TIMP-1 levels in healthy blood donors and 5 ng/ml of CEA
EXAMPLES
Example 1
Preparation of an ELISA to Quantitate Total TIMP-1 Concentrations
in Human Plasma
[0117] This example describes the preparation and validation of an
ELISA that measures total TIMP-1 levels in plasma. In addition,
this example provides information on TIMP-1 levels in different
plasma preparations as well as in healthy blood donors of both
sexes.
Materials and Methods:
Blood Donors
[0118] Blood samples were initially obtained from 94 apparently
healthy volunteer blood donors, comprising 51 males aged 19 to 59
years (median: 41 years) and 43 females aged 20 to 64 years
(median: 36 years). In a subsequent collection, 100 donor samples
were obtained, comprising 56 males aged 19 to 59 years (median 42:
years) and 44 females aged 20 to 60 years (median: 36.5 years).
Informed consent was obtained from all donors, and permission was
obtained from the local Ethical Committees.
Blood Collections and Plasma Separation
[0119] Peripheral blood was drawn with minimal stasis (if necessary
a maximum of 2 min stasis with a tourniquet at maximum +2 kPa was
acceptable) into pre-chilled citrate, EDTA, or heparin collection
tubes BECTON DICKINSON.RTM., Mountain View, Calif.), mixed 5 times
by inversion, and immediately chilled on ice. As soon as possible
(no later than 1.5 h after collection) the plasma and blood cells
were separated by centrifugation at 4.degree. C. at 1,200.times.g
for 30 min, and stored frozen at -80.degree. C. prior to assay.
Plasma pools were made with freshly collected samples from at least
ten donors, aliquoted and stored frozen at -80.degree. C. For
analysis, the samples were quickly thawed in a 37.degree. C. water
bath at and then placed on ice until needed.
Total TIMP-1 ELISA
[0120] A sensitive and specific sandwich ELISA was prepared, using
TIMP-1 antibodies developed at the Strangeways Laboratories (Hembry
et. al., 1985). A sheep polyclonal anti-TIMP-1 antiserum (Hembry
et. al., 1985; Murphy et. al., 1991) was used for antigen capture,
and a murine monoclonal anti-TIMP-1 IgG1 (MAC-15) (Cooksley et.
al., 1990) for antigen detection. A rabbit anti-mouse
immunoglobulin/alkaline phosphatase conjugate (Catalog number
D0314, DAKO.RTM., Glostrup, Denmark) was the secondary detection
reagent. The latter conjugate was supplied preabsorbed against
human IgG, thus eliminating cross-reactivity with IgG in the plasma
samples. As the monoclonal detection antibody MAC-15 recognises
both free TIMP-1 and TIMP-1 in complex with MMP's (Cooksley et.
al., 1990), the total TIMP-1 content captured by the sheep
polyclonal anti-TIMP-1 antiserum was quantitated by the ELISA.
[0121] 96-well microtiter plates (Maxisorp, Nunc, Roskilde,
Denmark) were coated for 1 h at 37.degree. C. with 100 .mu.L/well
of polyclonal sheep anti-TIMP-1 (4 mg/L) in 0.1 mol/L carbonate
buffer, pH 9.5. The wells were then rinsed twice with 200
.mu.L/well of SUPERBLOCK.RTM. J solution (Pierce Chemicals,
Rockford, Ill.) diluted 1:1 with phosphate-buffered saline (PBS).
The microtiter plates were stored for up to 14 days at -20.degree.
C. On the day of analysis, the plates were thawed at room
temperature and washed 5 times in PBS containing 1 g/L Tween.
[0122] A series of purified, recombinant human TIMP-1 standards
were used to calibrate each plate. Standards were prepared by
serially diluting a stock solution of purified TIMP-1. Standard
concentrations were 10, 5, 2.5, 1.25, 0.625, 0.313 and 0.156
.mu.g/L. Included on each plate was a blank containing only sample
dilution buffer, and 2 controls made from a 1:100 dilution of a
citrate plasma pool. One control was added as the first sample on
the plate and the second control was added as the last. All plasma
samples were diluted 1:100 in sample buffer consisting of 50 mol/L
phosphate, pH 7.2, 0.1 mol/L NaCl, 10 g/L bovine serum albumin
(Fraction V, Boehringer-Mannheim, Penzberg, Germany), and 1 g/L
Tween 20. A total of 100 .mu.L/well of each standard, blank,
control, and patient sample was incubated on the plate for 1 h at
30.degree. C. All standards, blanks, controls, and samples were run
in triplicate on each plate for every assay. After primary
incubation, the wells were washed 5 times, then treated for 1 h at
30.degree. C. with 100 .mu.L/well of purified MAC-15 monoclonal
antibody (0.5 mg/L) in sample dilution buffer. After another 5
washes the wells were incubated for 1 h at 30.degree. C. with 100
.mu.L/well of rabbit anti-mouse immunoglobulins(Ig)/alkaline
phosphatase conjugate diluted 1:2000 in sample dilution buffer.
Following 5 washes with washing solution and 3 washes with
distilled water, 100 .mu.L of freshly made p-nitrophenyl phosphate
(Sigma, St. Louis, Mo.) substrate solution (1.7 g/L in 0.1 mol/L
Tris.HCI, pH 9.5, 0.1 mol/L NaCl, 5 mmol/L MgCl.sub.2) was added to
each well. The plate was placed in a Ceres 900J plate reader
(BIO-TEK.RTM. Instruments, Winooski, Vt.) at 23.degree. C. with the
yellow color development automatically monitored. Readings were
taken at 405 nm against an air blank every 10 min. for one hour.
KinetiCalc II software was used to analyze the data by calculating
the rate of color formation for each well (linear regression
analysis), generating a 4-parameter fitted standard curve, and
calculating the TIMP-1 concentration of each plasma sample.
Recovery Experiments
[0123] The recovery of TIMP-1 signal was measured following
addition to 1:100 dilutions of citrate, EDTA or heparin plasma
pools. Purified TIMP-1 was added to plasma pools to give final
concentrations in the range of 0 to 10 .mu.g/L. The recovery in
each case was calculated from the slope of the line representing
TIMP-1 signal as a function of concentration, where 100% recovery
was defined as the slope obtained when TIMP-1 was diluted in sample
dilution buffer.
Immunoblotting
[0124] Citrate plasma from a patient with a high level of TIMP-1 in
blood (634 .mu.g/L, determined by ELISA), was diluted 1:10 and
added to a protein A-Sepharose column pre-incubated with polyclonal
sheep anti-TIMP-1. Following 5 cycles, bound proteins were eluted
from the column and 50 .mu.L of the resulting eluate run on 12%
SDS-gel electrophoresis READY GEL.RTM., BIO-RAD.RTM.). A mixture of
low molecular weight (Pharmacia) and high molecular weight markers
BIO-RAD.RTM.) and 50 .mu.L of TIMP-1 standard (100 .mu.g/L) in
Laemmli Sample Buffer were also run on the gel. Proteins were
transferred electrophoretically from the gel onto a polyvinylidene
difluoride (PVDF) membrane (MILLIPORE.RTM.). The membrane was
incubated for 1 h at room temperature with 1% skim milk powder in
TBS. Following washing, the membrane was incubated for 1 h at room
temperature with 20 ml of MAC-15 (5 mg/L). The membrane was then
washed and incubated for an additional hour at room temperature
with 20 ml of rabbit anti-mouse Ig/alkaline phosphatase conjugate
diluted 1:1000. Finally the membrane was washed and color developed
by the addition of a phosphate substrate solution (NBT/BCIP).
Results:
ELISA Performance
[0125] Development of color in each well progressed as a linear
function of time for all concentrations of total TIMP-1 in these
experiments (FIG. 1), with correlation coefficients for the fitted
lines typically greater than 0.99. The standard curve for the rates
plotted against the TIMP-1 concentration consisted of the linear
and upper curved regions (over the range of 0 to 5 .mu.g/L) of a
sigmoidal curve, and the correlation coefficient for the
4-parameter fit was typically better than 0.999 (FIG. 2). The rate
with no TIMP-1 (read against an air blank) was 1.21.+-.0.15
(mean.+-.SD) milliabsorbance units/min (n=29), while the rate with
10 .mu.g/L standard TIMP-1 was 50.3.+-.6.01 milliabsorbance
units/min (n=29). The limit of detection for the assay, defined as
the concentration of TIMP-1 corresponding to a signal 3 SD above
the mean for the TIMP-1 blank, was 0.089 .mu.g/L. This value was
13% of the mean of the measured concentrations of TIMP-1 in healthy
citrate plasma samples. The intra-assay coefficient of variation
(CV) for 16 replicates of a control citrate plasma pool was 5.3%,
and the inter-assay CV for 29 successive assays of the plasma pool
(run on different days) was 6.2%. This plasma pool had a TIMP-1
concentration of 57.8 .mu.g/L, corresponding to the 22nd centile of
the normal individuals.
[0126] Recovery of recombinant TIMP-1 after dilution in plasma
Specific recovery was determined by addition of increasing
concentrations of purified TIMP-1 to a panel of plasma pool
replicates, followed by subsequent measurement of signal. Recovery
was 104% in citrate plasma, 101% in diluted EDTA plasma, and 87% in
diluted heparin plasma (FIG. 3). Thus the recovery of TIMP-1 signal
from an internal standard was acceptable for all preparations of
plasma
Dilution Curves for Total Plasma TIMP-1 Signal
[0127] Serial dilutions of citrate, EDTA and heparin plasma pools
were made to test for linear reduction in TIMP-1 signal. Citrate,
EDTA and heparin plasmas all gave good linearity of signal as a
function of dilution. The 1% plasma dilution which was chosen for
subsequent determinations lay well within the range of this linear
dilution curve.
Immunoblotting of Total Plasma TIMP-1
[0128] A Western blot of an immunoabsorbed patient plasma sample
showed a clear band of 28 kDa (FIG. 4, lane 2), corresponding to
free, uncomplexed TIMP-1 (FIG. 4, lane 1). No bands were found at
the expected higher molecular weights corresponding to complexes
between MMP's and TIMP-1, e.g. MMP-2:TIMP-1, 100 kDa. This
indicates either that the major part of TIMP-1 was present in the
plasma as the free form, or that complexes were dissociated during
SDS-PAGE. Although the sample was left both unreduced and unheated
in order to preserve any complexes present in the plasma sample, it
has been reported that MMP:TIMP complexes may be unstable in
SDS-PAGE (Wilhelm et. al., 1989; Stetler-Stevenson et. al., 1989;
Moll et. al., 1990), even under non-reducing conditions (Moutsiakis
et. al., 1992).
Total TIMP-1 in Citrate and EDTA Plasma from the Same Healthy
Donor
[0129] A collection of citrate and EDTA plasma samples taken
simultaneously from 100 healthy donors was available for this
study. These samples were not specifically collected as
platelet-poor plasma. However, a small, representative number of
samples, prepared as platelet-poor plasma, did not differ
significantly in total TIMP-1 values. The percentile plots for
total TIMP-1 levels in these samples are shown in FIG. 5a. The
values in each set approximated a normal distribution. Citrate
plasma TIMP-1 levels ranged between 55.0 and 90.3 .mu.g/L (10th to
90th percentile) with a mean of 69.2.+-.13.1 .mu.g/L. Similarly,
EDTA plasma TIMP-1 levels ranged from 58.0 to 91.8 .mu.g/L with a
mean of 73.5.+-.14.2 .mu.g/L. For both citrate and EDTA plasma, the
mean TIMP-1 levels were in close proximity to the median levels
(Table 1). A paired means comparison showed that the level of
TIMP-1 in citrate plasma was significantly lower by 4.34 .mu.g/L
(95% CI 2.34-6.33; (p<0.0001) than the EDTA plasma level from
the same individual. However, it is likely that this difference may
be due to the variability in sampling procedure during plasma
collection. EDTA plasma tubes contained dry anticoagulant material,
while citrate plasma tubes contained a small amount of liquid
citrate buffer which gave a small and variable systematic dilution
error (.times. 9/10). The level of TIMP-1 in citrate plasma
correlated with that in EDTA plasma from the same individuals. The
linear regression plot in FIG. 5b shows a regression coefficient of
0.99 with a slope of the fitted line of 0.93, perhaps illustrating
this small dilution error. A non-parametric Spearman's rank test
for the data set gave a rho value of 0.62 and p<0.0001.
Total TIMP-1 Levels in Citrate Plasma
[0130] A total of 194 citrate plasma samples from healthy blood
donors were assayed, comprising 94 samples taken during one
collection and 100 samples taken 9 months later from a different
set of donors. FIG. 6 shows the percentile plots for TIMP-1 levels
measured in these two independent groups. The reference range for
TIMP-1 levels in citrate plasma from the first collection was 53.3
to 77.7 .mu.g/L (10th to 90th percentile) with a mean of
65.4.+-.10.1 .mu.g/L which was indistinguishable from the median
(Table 1). and approximating a normal distribution. The mean TIMP-1
level for the second collection was 69.2.+-.13.1 .mu.g/L (reference
range 55.0 to 90.3 .mu.g/L). An unpaired means comparison showed
that TIMP-1 levels in the two sets of samples taken during two
different periods differed only by 3.82 .mu.g/L (95% CI: 0.50-7.14
.mu.g/L; p=0.024). Moreover, no significant difference was apparent
between the controls (n=8) included in each set of assays (mean
difference 0.36 .mu.g/L; 95% CI: 1.71-2.44 .mu.g/L; p=0.69). The
mean TIMP-1 level for all 194 citrate plasma samples was
67.3.+-.11.8 .mu.g/L, close to the median of 66.1 .mu.g/L, with
levels again approximating a normal distribution (reference range
54.0 to 82.7 .mu.g/L).
TABLE-US-00001 TABLE 1 SUMMARY OF TOTAL TIMP-1 LEVELS DETERMINED IN
BLOOD FROM HEALTHY DONORS: Date of Number of Mean .+-. SD Median
Reference Blood fraction sampling samples (.mu.g/L) (.mu.g/L)
range* (.mu.g/L) Citrate plasma September 1996 94 65.4 .+-. 10.1
65.6 53.3-77.7 Citrate plasma May 1997 100** 69.2 .+-. 13.1 67.0
55.0-90.3 Citrate plasma 96 + 97 194 67.3 .+-. 11.8 66.1 54.0-82.7
EDTA plasma May 1997 100** 73.5 .+-. 14.2 71.2 58.0-91.8 *The
reference range is defined as the 10th to 90th percentile. **These
samples were collected from the same donors.
Tests for Correlations to Gender and Age of the Donor
[0131] In these studies, the control values measured in each assay
had a CV of 2.7%. Percentiles for TIMP-1 levels in 194 citrate
plasma samples calculated according to gender are shown in FIG. 7.
The mean TIMP-1 value for 107 male donors was 70.4.+-.12.0 .mu.g/L
(median 69.4 .mu.g/L) with a reference range from 56.2 to 86.6
.mu.g/L, while the mean TIMP-1 value for 87 female donors in this
set was 63.5.+-.10.5 .mu.g/L (median: 62.0 .mu.g/L) with a
reference range from 51.8 to 77.0 .mu.g/L. There was a significant
difference (p<0.0001) in TIMP-1 mean levels between the two
groups, with males having higher TIMP-1 levels than females. There
was a trend towards an increase in plasma TIMP-1 with increasing
age (Spearman's rho=0.33, P=0.0011), but this did not increase with
gender (females: Spearman's rho=0.29, P=0.006; males: Spearman's
rho=0.35, P=0.0003). In EDTA plasma, the mean TIMP-1 value for 56
males was 76.9.+-.15.0 .mu.g/L (median: 75.1 .mu.g/L) with a range
from 58.8 to 96.9 .mu.g/L, while 44 female donors had a mean TIMP-1
level of 69.3.+-.11.8 .mu.g/L (median: 67.9) with a reference range
from 56.1 to 85.5 .mu.g/L. Again, a significant difference appeared
between males and females (p=0.0076, unpaired means
comparison).
Discussion:
[0132] The assay described above enables accurate determination of
total TIMP-1 in human plasma samples. Kinetic rate assays of the
bound antigen were easily accomplished, permitting automated
fitting of rate curves, which has proven considerably more reliable
than single end-point measurements. The use of a rapid blocking
agent and a dilution buffer with high buffering capacity also
contributed to reproducible assays. Incorporating all these
elements in the final assay fulfilled the requirements of
sensitivity, specificity, stability, and good recovery of an
internal standard.
[0133] The quantitative studies in blood from healthy donors showed
that both citrate and EDTA plasma samples are suitable for TIMP-1
determination. Compared to other published studies of TIMP-1 i from
healthy donors (Jung et. al., 1996; Jung et. al., 1996), levels in
the present study fell within a very narrow range. Some studies
have reported results in serum, but plasma was selected for the
present study to avoid the variable contribution of platelet
activation to the measured TIMP-1 values (Cooper et. al., 1985).
While the plasma samples used in this study were not specifically
prepared as platelet-poor plasma, it was shown, based on tests
carried out in the lab, that this does not change the values. The
donor material was large enough to demonstrate that TIMP-1 levels
in healthy individuals (both EDTA and citrate) approximated a
normal distribution, for females as well as for males. Mean TIMP-1
levels were approx. 10% higher in males than in females for both
EDTA and citrate plasma. One explanation for this is a higher
release rate of TIMP-1 into blood from activated platelets,
reflecting a tendency towards higher incidence of thromboembolic
disease in the male population. When males and females were
considered separately, there was a weak correlation between TIMP-1
and age as seen for the whole population (see above).
Example 2
Preparation of an ELISA to Quantitate TIMP-1:MMP-9 Complexes in
Plasma
[0134] The following example describes an assay to determine the
concentration of TIMP-1:MMP-9 complexes in body fluids. The assay
is used with plasma samples of healthy blood donors in order to
establish normal ranges of this complex (Holten Andersen et. al.,
1999).
Materials and Methods:
TIMP-1: MMP-9 Complex ELISA
[0135] A sensitive and specific sandwich ELISA was prepared using
the above-described TIMP-1 antibody, MAC-15, and a rabbit MMP-9
polyclonal antibody developed in the Hematological Department,
Rigshospitalet, Denmark (Kjeldsen et. al., 1992). The MMP-9
antibody was used for antigen capture and MAC-15 was used for
antigen detection. A rabbit anti-mouse-Ig/alkaline phosphatase
conjugate (DAKO.RTM., Glostrup, Denmark) enabled a kinetic rate
assay (FIG. 8). The latter conjugate was supplied preabsorbed
against human IgG, thus eliminating cross-reactivity with IgG in
the plasma samples. The MMP-9 antibody captured both free MMP-9 and
MMP-9 complexed with TIMP-1, while MAC-15 only recognised TIMP-1.
Therefore only TIMP-1:MMP-9 complexes were quantitated by this
reagent pair.
[0136] To prevent spontaneous, ex vivo TIMP-1:MMP complex formation
during sampling and assay procedures, a protease inhibitor (i.e.
GALARDIN.RTM., Batimastat, Marimastat) was added to the plasma
sample after thawing. The addition of the protease inhibitor
blocked in vitro complex formation by inhibition of the catalytic
activity of the metalloproteinases.
[0137] The TIMP-1:MMP-9 assay was prepared and validated by a
method similar to that described above for the total TIMP-1 assay.
The TIMP-1:MMP-9 standard was prepared by incubating equimolar
amounts of purified recombinant TIMP-1 and MMP-9 (activated by
adding APMA) in PBS for 1 hour at 37 degrees Celsius.
[0138] Briefly, 96-well micotiter plates were coated overnight at
4.degree. C. with 100 .mu.L/well of rabbit polyclonal anti-MMP-9
antiserum in 0.1 mol/L carbonate buffer, pH 9.5. Prior to use,
assay wells were rinsed twice with 200 .mu.L/well of SuperBlock
solution diluted 1:1 with phosphate-buffered saline (PBS), and then
washed 5 times in PBS containing 1 g/L Tween 20. Wells were then
incubated for 1 h at 30.degree. C. with 100 .mu.L/well of plasma
diluted in sample buffer A series of purified TIMP-1:MMP-9
standards were used to calibrate each plate. Standards were
prepared by serially diluting a stock solution of purified
TIMP-1:MMP-9 complex. Included on each plate was a blank containing
only sample dilution buffer, and 2 controls made from a citrate
plasma pool. One control plasma pool was added as the first sample
on the plate and the second control was added as the last. All
standards, blanks, controls, and samples were run in triplicate on
each plate for every assay. After sample incubation and
TIMP-1:MMP-9 complex binding, the wells were washed 5 times,
followed by treatment for 1 h at 30.degree. C. with 100 .mu.L/well
of MAC-15 in sample dilution buffer. After another 5 washes, the
wells were incubated for 1 h at 30.degree. C. with 100 .mu.L/well
of rabbit anti-mouse Ig/alkaline phosphatase conjugate diluted in
sample dilution buffer. Following 5 washes with washing solution
and 3 additional washes with distilled water, 100 .mu.L of freshly
made p-nitrophenyl phosphate substrate solution was added to each
well. The plate was placed in a Ceres 900J plate reader at
23.degree. C. with the yellow color development monitored
automatically. Readings were taken at 405 nm against an air blank
every 10 min for one hour. KinetiCalc II software was used to
analyze the data by calculating the rate of color formation for
each well (linear regression analysis), generating a 4-parameter
fitted standard curve, and calculating the TIMP-1:MMP-9
concentration of each plasma sample.
Recovery Experiments
[0139] Specific recovery was determined by addition of TIMP-1:MMP-9
complex to a series of citrate, EDTA or heparin plasma pools. The
recovery in each case was calculated from the slope of the line
representing TIMP-1:MMP-9 complex signal as a function of
concentration, where 100% recovery was defined as the slope
obtained when TIMP-1:MMP-9 complex was diluted in the sample
dilution buffer.
Results
ELISA Performance
[0140] Development of color in each well was a linear function of
time for all concentrations of TIMP-1:MMP-9 complexes measured in
these experiments, with correlation coefficients for the
automatically fitted lines typically greater than 0.9. The
correlation coefficient for the 4-parameter fit was typically
greater than 0.999.
Recovery of TIMP-1:MMP-9 Complex after Dilution in Plasma
[0141] Specific recovery was determined by addition of increasing
concentrations of TIMP-1:MMP-9 to a plasma pool and subsequent
measurement of the specific signal.
Dilution Curves for Plasma TIMP-1:MMP-9
[0142] Serial dilutions of citrate and EDTA plasma pools were made
and complex levels quantitated to determine the linearity of the
assay. Citrate and EDTA plasmas all gave good linearity of signal
as a function of dilution.
Example 3
Preparation of an ELISA to Quantitate free TIMP-1 Levels in
Plasma
[0143] The following example describes an assay that determines the
concentration of free TIMP-1 levels in body fluids. The assay is
applied to plasma samples of healthy blood donors in order to
establish normal ranges of free TIMP-1.
Materials and Methods:
Free TIMP-1 ELISA
[0144] A sensitive and specific sandwich immunoassay was prepared,
using a TIMP-1 monoclonal IgG1 antibody (MAC-19) developed at the
Strangeways Laboratories, England (Cooksley et. al., 1990) and a
sheep polyclonal anti-TIMP-1 antibody. The sheep polyclonal
anti-TIMP-1 antibody was used for antigen capture and the murine
monoclonal MAC-19 was used for antigen detection. A rabbit
anti-mouse-Ig/alkaline phosphatase conjugate was the secondary
detection reagent (FIG. 9). The latter conjugate was supplied
preabsorbed against human IgG, thus eliminating cross-reactivity
with IgG in the plasma samples. The MAC-19 monoclonal antibody is
completely specific for free TIMP-1, which therefore is the only
form quantitated in this assay.
[0145] In order to test that MAC19 does not react with complexes
between TIMP-1 and MMP-9, the rabbit polyclonal anti-MMP-9 antibody
described in Example 2 was used for antigen capture and the mouse
monoclonal antibody MAC19 for antigen detection. A rabbit
anti-mouseIg/alkaline phosphatase conjugate was used as the
secondary labelled reagent. Standard TIMP-1:MMP-9 complex, free
MMP-9, free TIMP-1, and a blank control were assayed. FIG. 10 shows
that TIMP-1:MMP-9 complexes bound by the polyclonal anti-MMP-9
antibody are not detected by MAC19. An equivalent experiment was
performed, where MAC19 was substituted with MAC15. FIG. 11 shows
the results of this experiment. It is seen that MAC15 detects
TIMP-1:MMP-9 complex bound by the polyclonal anti-MMP-9
antibody.
[0146] To prevent ex vivo formation TIMP-1:MMP complexes during the
sampling and assay procedures, a protease inhibitor (i.e.
GALARDIN.RTM., Batimastat, Marimastat) can be added to the plasma
sample after thawing. The addition of the protease inhibitor will
prevent in vitro complex formation by competition with the
TIMP.
[0147] 96-well microtiter plates were coated for 1 h at 37.degree.
C. with 100 .mu.L/well of polyclonal sheep anti-TIMP-1 (4 mg/L) in
0.1 mol/L carbonate buffer, pH 9.5. The assay wells were then
rinsed twice with 200 .mu.L/well of SuperBlock solution diluted 1:1
with phosphate-buffered saline (PBS). The microtiter plates were
stored for up to 14 days at -20.degree. C. On the day of use, the
plates were thawed at room temperature and washed 5 times in PBS
containing 1 g/L Tween 20. Wells were then incubated for 1 h at
30.degree. C. with 100 .mu.L/well of triplicate 1:25 dilutions of
plasma made in a sample buffer consisting of 50 mol/L phosphate, pH
7.2, 0.1 mol/L NaCl, 10 g/L bovine serum albumin (Fraction V,
Boehringer-Mannheim, Penzberg, Germany) and 1 g/L Tween 20.
Standards were prepared by serially diluting a stock solution of
purified free TIMP-1 to yield concentrations of 10, 5, 2.5, 1.25,
0.625, 0.313 and 0.156 .mu.g/L. Included on each plate was a blank
containing only sample dilution buffer, and 2 controls made from a
citrate plasma pool diluted 1:25. One control plasma pool was added
as the first sample on the plate and the second control was added
as the last. All standards, blanks, controls, and samples were run
in triplicate on each plate for every assay. After TIMP-1 binding,
the wells were washed 5 times, then treated for 1 h at 30.degree.
C. with 100 .mu.L/well of the purified murine monoclonal anti-TIMP
MAC-19 (0.5 mg/L) in sample dilution buffer. After another 5 washes
the wells were incubated for 1 h at 30.degree. C. with 100
.mu.L/well of rabbit anti-mouse immunoglobulins/alkaline
phosphatase conjugate diluted 1:2000 in sample dilution buffer.
Following 5 washes with washing solution and 3 washes with
distilled water, 100 .mu.L of freshly made p-nitrophenyl phosphate
(Sigma, St. Louis, Mo.) substrate solution (1.7 g/L in 0.1 mol/L
Tris.HCI, pH 9.5, 0.1 mol/L NaCl, 5 mmol/L MgCl.sub.2) was added to
each well, and the plate was placed in a Ceres 900J plate reader
(BIO-TEK.RTM. Instruments, Winooski, Vt.) at 23.degree. C. The
yellow color development was monitored automatically, with readings
taken at 405 nm against an air blank every 10 min for one hour.
KinetiCalc II software was used to analyze the data, to calculate
the rate of color change for each well (linear regression
analysis), and to compute a 4-parameter fitted standard curve, from
which the free TIMP-1 concentration of each plasma sample was
calculated.
Dilution Curves and Recovery Experiments
[0148] These experiments were performed essentially as described in
Example 1, but using MAC19 instead of MAC15 for detection of free
TIMP-1 (as described above).
Healthy Donors
[0149] 108 donor plasma samples were obtained. Donors gave blood on
a volunteer basis and were all apparently healthy. Informed consent
was obtained from all blood donors, and permission was obtained
from the local Ethical Committees. The blood sampling and handling
were performed as described in Example 1.
Results
ELISA Performance
[0150] Development of color in each well was a linear function of
time for all concentrations of free TIMP-1 measured in these
experiments, with correlation coefficients for the automatically
fitted lines typically better than 0.9. The correlation coefficient
for the 4-parameter fit was typically better than 0.999. The
intra-assay variation for 24 triplicate measurements of the control
plasma pool was 9.6%.
Recovery of Recombinant TIMP-1 after Dilution in Plasma
[0151] Specific signal recovery was determined by addition of
increasing concentrations of purified TIMP-1 standard to a plasma
pool and subsequent measurement of the ELISA signal. In the diluted
citrate plasma pool 105% recovery was obtained, while 96% recovery
was obtained in the diluted EDTA plasma pool (FIG. 12).
Dilution Curves for Free Plasma TIMP-1 Signal
[0152] Serial dilutions of citrate and EDTA plasma pools were made
and free TIMP-1 levels assayed to test for linear reduction in
ELISA signal. Citrate and EDTA plasmas all gave good linearity of
signal as a function of dilution.
Healthy Blood Donors
[0153] Free TIMP-1 was measurable in all plasma samples. The median
free TIMP-1 concentration was 70.9 .mu.g/L (range: 32.3-169.7
.mu.g/L).
Discussion
[0154] This assay directly measures plasma free TIMP-1 levels. When
comparing free TIMP-1 levels with total TIMP-1 levels (the latter
measured with assay described in Example 1) in the 108 healthy
blood donors, a correlation coefficient of 0.46 (Rho, Spearman
Rank, p<0.0001) was obtained.
Example 4
Detection or Screening Value of Total TIMP-1 in Patients with
Colorectal Cancer
[0155] Total TIMP-1 levels in plasma from 591 colorectal cancer
patients (338 colon and 253 rectal) and in plasma from 108 age and
gender matched healthy individuals were measured with the TIMP-1
assay described in Example 1. The TIMP-1 values were analyzed and
compared using standard biostatistical parameters.
Materials and Methods
Patients
[0156] 591 patients undergoing elective surgery for pathologically
confirmed colorectal cancer were included in the study. Blood
samples were obtained preoperatively with informed consent from all
patients in accordance with the Helsinki declaration, and
permission was granted by the local ethical committee of Hvidovre
Hospital, Denmark. All patients had pathologically verified
adenocarcinoma of the colon or rectum. It was found that 59 (10%)
patients could be classified as having Dukes' stage A disease, 219
(37%) patients Dukes' stage B, 170 (29%) patients Dukes' stage C
and 143 (24%) patients Dukes' stage D. 338 tumors were colon
cancers and 253 were rectal cancers. Clinical data such as age, sex
and survival after surgery were collected. The median age of the
patients was 69 years (range 33-90 years) with 237 females and 354
males represented in the patient cohort.
[0157] A second patient cohort was collected prospectively. This
cohort consisted of 21 rectal cancer and 43 colon cancer patients.
There were 11 patients with Dukes' stage A, 27 with Dukes' stage B,
14 with Dukes' stage C, and 13 with Dukes' stage D disease.
Healthy Donors
[0158] The same donor population as described in Example 3.
Blood Samples
[0159] Blood samples (5 ml) were collected preoperatively from all
patients on the day of their surgery. To ensure valid TIMP-1
measurements, peripheral blood was drawn with minimal stasis and
collected in EDTA anticoagulant tubes (BECTON-DICKINSON.RTM.,
Mountain View, Calif.) in accordance with a previously described
protocol (Example 1).
TIMP-1 ELISA
[0160] TIMP-1 levels were measured in EDTA plasma samples using the
assay described in Example 1.
Results:
Total TIMP-1 Levels in Plasma
[0161] Using a kinetic rate assay, total TIMP-1 levels were
determined in all patient and healthy donor plasma samples. Every
plasma sample had measurable levels of TIMP-1, with a median total
TIMP-1 value for the 591 colorectal cancer patients of 141.1
.mu.g/L (range 53.7-788.7 .mu.g/L). When stratified into colon and
rectal cancer, the median values were 158.6 .mu.g/L (range:
53.7-788.7 .mu.g/L) for colon and 126.3 (range: 64.1-640.1 .mu.g/L)
for rectal cancer. There was a statistically significant difference
in TIMP-1 levels when the patient material was stratified according
to Duke's stage, with Dukes' A being the lowest and Dukes' D the
highest (Kruskal-Wallis test, P<0.0001). However, the highest
TIMP-1 levels were not restricted to advanced disease, and no
significant difference in total plasma TIMP-1 levels was seen among
patients with Dukes A-C disease. A relatively weak correlation
between plasma TIMP-1 and age was found (r=0.35; p<0.0001).
There was no significant difference in TIMP-1 levels between males
and females (p=0.97).
[0162] The median TIMP-1 level in plasma from healthy donors was
88.6 .mu.g/L with a range of 51.0-156.2 .mu.g/L. There was a highly
significant statistical difference in the total plasma TIMP-1
values between the colorectal cancer patients and the healthy blood
donors.
[0163] The median total TIMP-1 value for the 64 colorectal cancer
patients was 138.2 .mu.g/L (range: 80.7-790.6 .mu.g/L). Stratifying
the patients into colon and rectal cancer, the median, total TIMP-1
values were 152.2 .mu.g/L (range: 80.7-626.2 .mu.g/L) for colon and
133.6 (range: 84.3-790.6) for rectal cancer. There was a highly
significant statistical difference in the total plasma TIMP-1
values between the colon and rectal cancer patients each compared
with the healthy blood donors.
Detection or Screening Value of Total TIMP-1
[0164] Using the measured total TIMP-1 levels in plasma from
healthy donors and the 591 colorectal cancer patients, Receiver
Operating Characteristics (ROC) curves were generated to evaluate
the detection or screening value of total TIMP-1. As seen in FIG.
13, the ROC curve was initially steep, indicating a high
sensitivity and specificity of total TIMP-1 as a marker for
colorectal cancer. It appears that the AUC is greater for colon
cancer than for rectal cancer. FIG. 14 shows a similar ROC curve
now including only patients with early stage colorectal cancer,
i.e. Dukes' stage A or B disease. Also shown is the ROC curve for
early stage (Dukes' stage A and B) right-sided colon cancer.
[0165] Using the total TIMP-1 levels in plasma from healthy donors
and in the second cohort of 64 colorectal cancer patients, ROC
curves were again constructed to confirm the detection or screening
value of TIMP-1. As seen in FIG. 15, the curve was again initially
steep, indicating a high sensitivity and specificity of total
TIMP-1 as a marker for colorectal cancer.
[0166] An additional study of 180 healthy blood donors and 20
colorectal cancer patients, using different antibodies (Anti TIMP-1
11E/C6, Anti-TIMP-1 RRU-T6) (the hybridomas producing these two
antibodies were deposited Apr. 10, 2000 with ATCC) in an automated
immunoassay, further corroborated the previous clinical results.
Moreover, the absolute values generated from the automated assay
showed a high degree of correlation to those obtained by the assay
described in Example 1 (r=0.9).
Discussion:
[0167] These data suggest that total TIMP-1 measurements in plasma
can be used as a detection or screening procedure to aid in
identifying patients with a high risk of having colorectal cancer.
In particular, total TIMP-1 was as effective in identifying
patients with early cancer (Duke's stage A+B) as identifying
patients with more advanced disease. Also, total TIMP-1 was even
more effective in identifying patients with early stage,
right-sided colon cancer, a procedure that is difficult with
conventional detection or screening procedures. Right-sided colon
cancer cannot be visualized by flexible sigmoidoscopy, a standard
colon cancer screening methodology. It has a more insidious onset
than do left-sided lesions, and clinical symptoms develop only in
late stage disease. Early detection or screening of right sided
colon cancer has the potential to reduce the mortality of this
disease.
[0168] Moreover, the smaller, prospective trial corroborated the
results of the larger retrospective study, further confirming the
detection or screening value of total TIMP-1 in patients suffering
from colorectal cancer.
Example 5
Quantitation of Total TIMP-1 in Plasma from Patients with
Inflammatory Bowel Diseases
Patients
[0169] 46 patients with IBD (Inflammatory Bowel Disease) were
included in the study. 22 patients had ulcerative colitis and 24
patients had Crohn's Disease. Total TIMP-1 levels in EDTA plasma
from healthy blood donors and colorectal cancer patients (Examples
3 and 4) were included for comparison.
Total TIMP-1 Values
[0170] Total TIMP-1 levels were measured in the EDTA plasma samples
using the sandwich assay described in Example 1.
Results:
[0171] The measured total TIMP-1 values are shown in Table 2.
TABLE-US-00002 TABLE 2 Ulcerative Crohn's IBD colitis disease total
n = 22 n = 24 n = 46 Median total TIMP-1 (.mu.g/L) 79.5 78.3 84.8
Range total TIMP-1 (.mu.g/L) 54.9-189.9 49.0-156.2 38.7-154.5
Healthy Colorectal donors Cancer n = 108 n = 591 Median total
TIMP-1 (.mu.g/L) 89.1 141 Range total TIMP-1 (.mu.g/L) 51.0-156.2
53.7-789
[0172] There was no significant difference when total TIMP-1 values
from patients with IBD and healthy blood donors were compared
(Mann-Whitney; p=0.45). There was a highly significant difference
between total plasma TIMP-1 levels between patients with IBD and
the 591 colorectal cancer patients (Mann-Whitney; p<0.0001). A
graphical representation of these results is depicted in FIG.
16.
Discussion:
[0173] These results demonstrate that patients with colorectal
cancer have significantly higher total TIMP-1 plasma levels than do
patients with IBD. Moreover, patients with IBD had total TIMP-1
levels equivalent to those found in healthy blood donors, showing
that plasma total TIMP-1 can be used as a highly sensitive and
specific marker to distinguish between non-malignant and malignant
diseases of the gastrointestinal tract.
Example 6
Detection or Screening Value of Total TIMP-1 in Combination with
CEA in Patients with Colorectal Cancer
[0174] Total TIMP-1 in plasma from 591 colorectal cancer patients
(338 colon and 253 rectal) and in plasma from 108 age and gender
matched healthy individuals was measured using the TIMP-1 assay
described in Example 1. In addition, CEA was measured in the
corresponding patient and donor serum samples using a commercially
available, chemiluminescent CEA EIA kit (IMMULITE.RTM. CEA,
DPC.TM., Los Angeles, Calif., USA). The TIMP-1 and CEA values from
healthy donors and cancer patients were combined by logistic
regression analysis and ROC curves were generated.
Results:
Detection or Screening Value of Total TIMP-1 and CEA
[0175] Calculating the sensitivity and specificity of CEA in the
591 colorectal cancer patients when including the 108 healthy
donors a cut-off providing 98% specificity gave a sensitivity of
35%. When stratifying patients into colon or rectal cancer, the
sensitivity was 37% and 33%, respectively at the same level of
specificity. Including only patients with right-sided colon cancer,
it is demonstrated in FIG. 17 that the sensitivity increased to
45%.
[0176] When the total TIMP-1 values from Example 4 are included
together with CEA, the sensitivity of the marker combination was
found by logistic regression analysis to be 52%. The additional
sensitivity obtained by the addition of CEA measurements in serum
is highly significant (p<0.0001). When stratifying the patient
cohort into colon and rectal cancer, the sensitivity was 61% and
39%, respectively at the 98% specificity level. Including only
patients with right-sided colon cancer, the sensitivity was 74%. A
graphical illustration of these results appears from FIG. 17.
Discussion:
[0177] These data show that by adding an additional marker, an
improvement in the sensitivity of total TIMP-1 can be obtained,
while maintaining a high specificity of 98%. Thus, the combination
of CEA and TIMP-1 could be useful as a detection or screening
procedure to identify patients with a high risk of having
colorectal cancer. In particular, this combination was efficient in
identifying patients with early stage cancer (Dukes' stage A+B).
Also, this combination was highly effective in identifying patients
with early stage, right-sided colon cancer.
Example 7
Lack of Detection or Screening Value of Plasma Free TIMP-1 Levels
in Patients with Colorectal cancer
Materials and Methods
[0178] Free TIMP-1 levels in plasma from 64 colorectal cancer
patients (43 colon and 21 rectal) and in plasma from 108 age
matched, healthy individuals were measured using the TIMP-1 assay
described in Example 3. The free TIMP-1 values were analysed and
compared using standard biostatistical parameters.
Results:
Free TIMP-1 Levels in Plasma
[0179] Using the kinetic rate ELISA described in Example 3, free
TIMP-1 levels were measured in all patient and healthy donor plasma
samples. All samples had measurable levels of free TIMP-1, with a
median free TIMP-1 value of 82.0 .mu.g/L (range: 44.7-424.0
.mu.g/L) for the colorectal cancer patients. The median free TIMP-1
level in plasma from healthy donors was 70.9 .mu.g/L, with a range
of 32.3-169.7 .mu.g/L. While no significant difference in free
TIMP-1 levels was found among patients with Dukes' stage A-C
disease, patients with Dukes' stage D had significantly elevated
free plasma TIMP-1 levels compared to the patients with Dukes'
stage A and B disease. When comparing total TIMP-1 values with free
TIMP-1 values in plasma from these 64 colorectal cancer patients a
correlation coefficient of 0.91 (Rho, Spearman Rank, p<0.0001)
was found.
Lack of Detection or Screening Value of Free TIMP-1
[0180] FIG. 18 shows the ROC curves generated from the plasma
measurements of free TIMP-1. The AUC is 0.61 when determining the
detection or screening performance of free TIMP-1.
Discussion:
[0181] These data show that free TIMP-1 alone is not likely to be
useful as a screening or detection marker to identify patients with
a high risk of having colorectal cancer.
Example 8
Detection or Screening Value of TIMP-1:MMP-9 Complex Measurements
in Patients with Colorectal Cancer
[0182] TIMP-1:MMP-9 complex levels in plasma from colorectal cancer
patients and in plasma from age and gender matched healthy
individuals can be measured using the TIMP-1:MMP-9 assay described
in Example 2. TIMP-1:MMP-9 complex values from healthy donors and
cancer patients can be compared and the detection or screening
value determined.
[0183] Using the measured values of free, total, and TIMP-1:MMP-9
complex levels, the detection or screening value of ratios or
fractions can be calculated. In addition other molecules e.g. serum
CEA can be added to these calculations to generate mathematical
algorithms to increase the overall detection or screening
value.
Example 9
Detection or Screening Value of the Combination of Plasma Total
TIMP-1 and Plasma Free TIMP-1 in Patients with Colorectal
Cancer
[0184] Total TIMP-1 levels in plasma from 64 colorectal cancer
patients and in plasma from 108 age and gender matched healthy
individuals were measured with the TIMP-1 assay described in
Example 1. Using the assay described in Example 3, plasma free
TIMP-1 levels were measured in the same individuals as described
above. The total and the free TIMP-1 values were analyzed and
compared using logistic regression analysis.
Materials and Methods:
Patients
[0185] 64 patients undergoing elective surgery for pathologically
confirmed colorectal cancer were included in the study. This cohort
consisted of 21 rectal cancer and 43 colon cancer patients. There
were 11 patients with Dukes' stage A, 27 with Dukes' stage B, 14
with Dukes' stage C, and 13 with Dukes' stage D disease. Blood
samples were obtained preoperatively with informed consent from all
patients in accordance with the Helsinki declaration, and
permission was granted by the local ethical committee of Hvidovre
Hospital, Denmark. All patients had pathologically verified
adenocarcinoma of the colon or rectum. Clinical data such as age,
sex and survival after surgery were collected.
Healthy Donors
[0186] The same donor population as described in Example 3.
Blood Samples
[0187] Blood samples (5 mL) were collected preoperatively from all
patients on the day of their surgery. To ensure valid TIMP-1
measurements, peripheral blood was drawn with minimal stasis and
collected in EDTA anticoagulant tubes (BECTON-DICKINSON.RTM.,
Mountain View, Calif.) in accordance with a previously described
protocol (Example 1).
Total and Free TIMP-1 Plasma Measurements
[0188] TIMP-1 levels were measured in EDTA plasma samples using the
assays described in Example 1 and Example 3.
Results:
TIMP-1 Levels in Plasma
[0189] The total TIMP-1 levels in these 64 patients with colorectal
cancer are described in Example 4, and the free TIMP-1 levels in
these patients are described in Example 7.
[0190] Using the total and the free TIMP-1 levels in plasma from
healthy donors and from the 64 colorectal cancer patients, ROC
curves were constructed for each of these TIMP-1 forms. The total
TIMP-1 values obtained confirm the detection or screening value of
total TIMP-1 measurements in patients with colorectal cancer. As
seen in FIG. 15, the curve was again initially steep, indicating a
high sensitivity and specificity of total TIMP-1 as a marker for
colorectal cancer. The ROC curve for free TIMP-1 was discussed in
Example 7.
[0191] The corresponding data for total TIMP-1 in this patient
population gave an AUC of 0.88. However, an increase in the
detection or screening alue was obtained when combining free and
total TIMP-1 (AUC=0.94). This increase is highly statistically
significant, p<0.0001 and shows the value of the important
embodiment of using both free and total TIMP-1 in the same
analysis.
Discussion:
[0192] These data suggest that the combination of total TIMP-1 and
free TIMP-1 measurements in plasma can be used as a screening or
detection procedure to aid in identifying patients with a high risk
of having colorectal cancer.
[0193] In a similar manner as described in the present example,
other ratios and/or combinations or mathematical permutations
between free and total TIMP-1 values can be calculated and used for
detection or screening purposes.
[0194] Calculations of relationships among all the various forms of
TIMP-1, including total and free TIMP-1, total concentration of
complexes (total TIMP-1-free TIMP-1), and/or the concentration of
TIMP-1:MMP-9, might be extremely useful in the management, and
especially distinguishing of patients with non-malignant diseases
from patients with cancer.
Example 10
Lack of Detection or Screening Value of Total TIMP-1 Measurements
in Patients with Primary (Stage I and II) Breast Cancer
Materials and Methods
[0195] Using the total TIMP-1 assay described in Example 1,
pre-operative plasma samples from 322 patients with primary breast
cancer were analysed for total TIMP-1 content. In addition, 108
plasma samples from healthy blood donors were evaluated. The median
total TIMP-1 for the breast cancer patients was 88.3 .mu.g/L
(range: 45.5-289.3 .mu.g/L), while the median total TIMP-1 level
for the healthy blood donors was 88.9 .mu.g/L (range: 51.0-156.2
.mu.g/L). There was no statistical significant difference in total
TIMP-1 plasma levels between the two groups (Mann-Whitney, p=0.87).
FIG. 19 shows a ROC-curve including the above mentioned data.
Discussion
[0196] These data demonstrate that patients with primary breast
cancer have total TIMP-1 values that are not significantly
different from those of healthy blood donors. Thus, these data
support the specificity of TIMP-1 measurements in the detection or
screening of patients with colorectal cancer.
Example 11
[0197] Detection or screening value of TIMP-2 in patients with
colorectal cancer.
[0198] This example describes total TIMP-1 determinations from
patients with stage IV breast cancer.
Materials and Methods:
Patients and Blood Donors
[0199] Blood was collected from 19 stage IV breast cancer patients
(aged 45 to 70 years) at the Oncology Department, Herlev University
Hospital, Copenhagen, Denmark, and from 87 healthy female blood
donors (Example 1).
Total TIMP-1 Plasma Levels
[0200] Total TIMP-1 levels were measured in all EDTA plasma samples
using the assay described in Example 1.
Results:
[0201] Total TIMP-1 levels in plasma from patients with advanced
breast cancer Total TIMP-1 was measured in EDTA plasma samples from
19 breast cancer patients with stage IV disease. These levels were
compared with total TIMP-1 levels in 87 healthy female donors. The
mean, total TIMP-1 level measured in the 19 breast cancer patients
was 292.+-.331 .mu.g/L (median: 236 .mu.g/L), compared with a mean,
total TIMP-1 level of 63.5.+-.10.5 .mu.g/L (median: 62.0 .mu.g/L)
in 87 healthy female donors. A Wald-Wolfowitz runs test indicated a
highly significant difference (p<0.0001) between patient total
TIMP-1 levels and those of healthy donors. FIG. 20 shows a
percentile plot of total TIMP-1 levels in from the 19 breast cancer
patients and the TIMP-1 levels found in plasma from the 87 healthy
female donors.
Discussion:
[0202] These data show that plasma TIMP-1 measurements can be used
to monitor breast cancer patients for recurrence of disease.
Example 12
Diagnostic Value of TIMP-2 Measurements in Patients with Colorectal
Cancer
Materials and Methods
[0203] TIMP-2 levels were measured with an in-house TIMP-2 assay in
plasma from 64 colorectal cancer patients (43 colon and 21 rectal)
and in plasma from 108 age matched healthy individuals. The
measured TIMP-2 values from healthy donors and cancer patients were
compared.
Results:
[0204] TIMP-2 was measurable in all samples. No significant
differences in TIMP-2 levels were found between healthy blood
donors and colorectal cancer patients.
Discussion
[0205] These data support the specificity of TIMP-1 measurements in
the detection or screening of patients with colorectal cancer,
supporting the unique value of TIMP-1 as an aid in the early
detection of colorectal cancer.
Example 13
Total TIMP-1 as Obtained 6 Months Post-Operatively in Patients with
a Prior Surgery for Colorectal Cancer
[0206] Total TIMP-1 levels in plasma obtained 6 months
post-operatively from 317 colorectal cancer patients and in plasma
from 108 age and gender matched healthy individuals were measured
with the TIMP-1 assay described in Example 1. The TIMP-1 values
were analyzed and compared using standard biostatistical
parameters.
Materials and Methods:
Patients
[0207] 317 colorectal cancer patients were included in the study.
48 patients had Dukes' A disease, 141 Dukes' B, 120 Dukes' C and 9
Dukes' D disease. All patients received intended curative surgery
but no adjuvant chemotherapy was administered. Corresponding plasma
and serum samples were obtained preoperatively and 6 months
postoperatively with informed consent from all patients in
accordance with the Helsinki declaration, and permission was
granted by the local ethical committee of Hvidovre Hospital,
Denmark. The median follow-up time was 82 months (range 68 to 95);
152 patients died, 69 patients experienced local recurrence and 54
patients developed distant metastases during the observation
period.
Healthy Donors
[0208] The same donor population as described in Example 3.
Blood Samples
[0209] Blood samples (5 ml) were collected post-operatively from
all patients. To ensure valid TIMP-1 measurements, peripheral blood
was drawn with minimal stasis and collected in EDTA anti-coagulant
tubes (BECTON-DICKINSON.RTM., Mountain View, Calif.) in accordance
with a previously described protocol (Example 1).
TIMP-1 ELISA
[0210] TIMP-1 levels were measured in EDTA plasma samples using the
assay described in Example 1.
Results:
Total TIMP-1 Levels in Plasma
[0211] Using a kinetic rate assay, total TIMP-1 levels were
determined in all patient and healthy donor plasma samples. Every
plasma sample had measurable levels of TIMP-1.
[0212] The median TIMP-1 level in plasma from healthy donors was
88.6 .mu.g/L with a range of 51.0-156.2 .mu.g/L. There was a highly
significant statistical difference in the total plasma TIMP-1
values between the colorectal cancer patients and the healthy blood
donors FIG. 21.
[0213] The 95.sup.th percentile of the healthy blood donors was 134
.mu.g/L plasma
Example 14
Detection or Screening Value of Total TIMP-1
[0214] Total TIMP-1 levels in plasma obtained preoperatively and 6
months post-operatively from 309 colorectal cancer patients with
Dukes A+B cancer or Dukes C cancer. Plasma TIMP-1 was measured in
samples obtained preoperatively and 6 months post-operatively.
Plasma TIMP-1 was measured with the TIMP-1 assay described in
Example 1. The TIMP-1 values were analyzed and compared using
standard biostatistical parameters.
Materials and Methods:
Patients
[0215] 309 colorectal cancer patients were included in the study.
48 patients had Dukes' A disease, 141 Dukes' B, and 120 Dukes' C.
All patients received intended curative surgery but no adjuvant
chemotherapy was administered. Corresponding plasma and serum
samples were obtained preoperatively and 6 months postoperatively
with informed consent from all patients in accordance with the
Helsinki declaration, and permission was granted by the local
ethical committee of Hvidovre Hospital, Denmark. The median
follow-up time was 82 months (range 68 to 95)
Blood Samples
[0216] Blood samples (5 ml) were collected post-operatively from
all patients. To ensure valid TIMP-1 measurements, peripheral blood
was drawn with minimal stasis and collected in EDTA anti-coagulant
tubes (BECTON-DICKINSON.RTM., Mountain View, Calif.) in accordance
with a previously described protocol (Example 1).
TIMP-1 ELISA
[0217] TIMP-1 levels were measured in EDTA plasma samples using the
assay described in Example 1. Plasma TIMP-1 levels were scored as
low or high based on the 95th percentile of plasma TIMP-1 in an age
matched healthy control group.
Results
[0218] Preoperative and postoperative plasma TIMP-1 levels were
scored as low or high based on the 95th percentile of plasma TIMP-1
in an age matched healthy control group. 85% (181/214) of the
patients with low preoperative TIMP-1 levels remained low
postoperatively (group I) and accordingly 15% (33/214) progressed
to high postoperative TIMP-1 levels (group III). 53% (55/103) of
the patients with high preoperative levels had low postoperative
TIMP-1 (group II) and accordingly 47% (48/103) remained high 6
months following surgery (group IV). Analysis of overall survival
showed a significant difference between groups (p<0.0001). With
group I as the baseline, group II had a hazard ratio (HR) of 1.1
(95% CI:0.7 to 1.7), group III 2.0 (1.2 to 3.3) and group IV 2.7
(1.8 to 4.0). Multivariate analysis including Dukes' stage, age,
gender, tumor location, serum CEA and plasma TIMP-1 (as grouped
into the above mentioned four groups), showed that plasma TIMP-1
was a significant (p=0.0004) predictor of survival. Similar results
were obtained for time to local recurrence and detection of distant
metastases. Separate analyses including only the 6 month value of
TIMP-1 indicated that elevated levels were associated with shorter
survival (p<0.0001) (FIG. 22).
Discussion:
[0219] These data suggest that comparing preoperative and
postoperative total TIMP-1 measurements in plasma can be used as a
monitoring procedure to aid in identifying patients with a high
risk of having MRD and/or recurrent colorectal cancer.
Example 15
Detection or Screening Value of Total TIMP-1 as Obtained 6 Months
Post-Operatively in Patients with a Prior Surgery for Dukes' A+B or
C Colorectal Cancer
[0220] Total TIMP-1 levels in plasma obtained 6 months
post-operatively from 309 colorectal cancer patients with Dukes A+B
cancer or Dukes C cancer. Plasma TIMP-1 was measured in samples
obtained 6 months post-operatively. Plasma TIMP-1 was measured with
the TIMP-1 assay described in Example 1. The TIMP-1 values were
analyzed and compared using standard biostatistical parameters.
Materials and Methods:
Patients
[0221] 309 colorectal cancer patients were included in the study.
48 patients had Dukes' A disease, 141 Dukes' B, and 120 Dukes' C.
All patients received intended curative surgery but no adjuvant
chemotherapy was administered. Corresponding plasma and serum
samples were obtained 6 months postoperatively with informed
consent from all patients in accordance with the Helsinki
declaration, and permission was granted by the local ethical
committee of Hvidovre Hospital, Denmark. The median follow-up time
was 82 months (range 68 to 95)
Blood Samples
[0222] Blood samples (5 ml) were collected post-operatively from
all patients. To ensure valid TIMP-1 measurements, peripheral blood
was drawn with minimal stasis and collected in EDTA anticoagulant
tubes (BECTON DICKINSON.RTM., Mountain View, Calif.) in accordance
with a previously described protocol (Example 1).
TIMP-1 ELISA
[0223] TIMP-1 levels were measured in EDTA plasma samples using the
assay described in Example 1. Postoperative plasma TIMP-1 levels
were scored as low or high based on the 95th percentile of plasma
TIMP-1 in an age matched healthy control group.
Results:
Total TIMP-1 Levels in Plasma
[0224] Using a kinetic rate assay, total TIMP-1 levels were
determined in all patient plasma samples. Every plasma sample had
measurable levels of TIMP-1.
Detection or Screening Value of Total TIMP-1
[0225] Using the measured total TIMP-1 levels in plasma from the
309 colorectal cancer patients, univariate survival analyses was
performed. As seen in FIG. 23, Dukes A+B patients with low
post-operative plasma TIMP-1 levels had a significantly better
survival than Dukes A+B patients with high post-operative plasma
TIMP-1 levels. Similar results were obtained in patients who prior
to the blood sampling underwent surgery for Dukes C disease. It is
noteworthy, that Dukes A+B patients with high post-operative plasma
TIMP-1 levels had a survival similar to the survival of Dukes C
patients with low post-operative TIMP-1 plasma levels.
Discussion:
[0226] These data suggest that total post-operative TIMP-1
measurements in plasma can be used as a monitoring procedure to aid
in identifying Dukes A, B and C patients with a high risk of having
MRD and/or recurrent colorectal cancer.
Example 16
Detection or Screening Value of Total TIMP-1 in Combination with
CEA in Patients with a Prior Surgery for Colorectal Cancer
[0227] Total TIMP-1 in plasma from 317 colorectal cancer patients
was measured using the TIMP-1 assay described in Example 1. In
addition, CEA was measured in the corresponding patient serum
samples using a commercially available, chemiluminescent CEA EIA
kit (IMMULITE.RTM.CEA, DPC.TM., Los Angeles, Calif., USA). The
TIMP-1 and CEA values from the cancer patients were used to
construct univariate survival curves (metastasis free survival
(MFS) and overall survival (OS)). The patients were dichotomised
using the 95.sup.th percentile plasma TIMP-1 level of a healthy
blood donor population.
Results:
Detection or Screening Value of Total TIMP-1 and CEA
[0228] As seen from FIG. 24 and FIG. 25, patients with low TIMP-1
and low CEA levels had a MFS and OS which was significantly better
than those of patients with one or both parameters being high. On
the other hand, patients with both parameters high had a MFS and OS
which were significantly worse than those of patients with one or
none of the parameters being high (FIG. 24 and FIG. 25).
Discussion:
[0229] These data show that by adding an additional marker (CEA),
an improvement in the monitoring value of total TIMP-1 can be
obtained. Thus, the combination of CEA and TIMP-1 could be useful
as a monitoring procedure to identify patients with a high risk of
having MRD or recurrent colorectal cancer. It should be noted that
this combination was efficient in identifying patients with early
stage cancer (Duke's stage A+B)' with a high probability of MRD
and/or recurrent colorectal cancer.
Example 17
Quantitation of Total TIMP-1 in Plasma from Patients with
Inflammatory Bowel Diseases
Patients
[0230] 46 patients with IBD (Inflammatory Bowel Disease) were
included in the study. 22 patients had ulcerative colitis and 24
patients had Crohn's Disease. Total TIMP-1 levels in EDTA plasma
from healthy blood donors and post-operatively in colorectal cancer
patients (Examples 4) were included for comparison.
Total TIMP-1 Values
[0231] Total TIMP-1 levels were measured in the EDTA plasma samples
using the sandwich assay described in Example 1.
Results:
[0232] The measured total TIMP-1 values are shown in Table 2.
TABLE-US-00003 TABLE 2 Ulcerative Crohn's IBD colitis disease total
N = 22 n = 24 n = 46 Median total TIMP-1 (.mu.g/L) 79.5 78.3 84.8
Range total TIMP-1 (.mu.g/L) 54.9-189.9 49.0-156.2 38.7-154.5
Healthy Colorectal donors Cancer n = 108 n = 315 Median total
TIMP-1 (.mu.g/L) 89.1 105 Range total TIMP-1 (.mu.g/L) 51.0-156.2
44-450
[0233] There was no significant difference when total TIMP-1 values
from patients with IBD and healthy blood donors were compared
(Mann-Whitney; p=0.45). There was a highly significant difference
between total plasma TIMP-1 levels between patients with IBD and
the 315 colorectal cancer patients (Mann-Whitney; p<0.0001).
Discussion:
[0234] These results demonstrate that patients with colorectal
cancer have significantly higher total TIMP-1 plasma levels than do
patients with IBD. Moreover, patients with IBD had total TIMP-1
levels equivalent to those found in healthy blood donors, showing
that plasma total TIMP-1 can be used as a highly sensitive and
specific marker to distinguish between non-malignant and malignant
diseases of the gastrointestinal tract.
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