Note: Descriptions are shown in the official language in which they were submitted.
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DETECTION OF FIBRIN AND FIBRINOGEN DEGRADATION PRODUCTS
AND ASSOCIATED METHODS OF PRODUCTION AND USE FOR THE
DETECTION AND MONITORING OF CANCER
FIELD OF THE INVENTION
[0001] The present application relates generally to methods of producing and
to the
production of antibody populations against fibrinogen and fibrin degradation
products
(FDP), to the antibody populations themselves and to related methods of use,
to the
detection of cancers and for monitoring the progress of cancer treatment by
immunochemically measuring the quantity of FDP in serum.
BACKGROUND OF THE INVENTION
[0002] Despite recent advances in the understanding of cancer, current
techniques
for the screening and identification of cancer leave room for improvement.
Methods
known in the art for screening cancer attempt to detect cancer-related
antigens by using
antibodies. Antigens are macromolecules, such as proteins, nucleic acids or
polysaccharides, which are capable of eliciting an immune response in the
body. The
immune systems of mammals and other animals have the ability to detect foreign
agents, such as the antigens associated with cancer, and to respond to these
antigens
by producing antibodies which specifically target and react with those cancer-
associated
antigens. Thus, there is a strong correlation between the detection of these
cancer-
associated antigens or the circulating antibodies that target these antigens
in a
mammal's circulating blood and the existence of cancer in that individual. As
a result,
tests that detect the existence of such antigens or antibodies are useful in
the screening
and diagnosis of cancer.
[0003] One promising cancer-associated antigen target is a pool comprised of
both
fibrin and fibrinogen degradation products (FDP). While the production of both
fibrin
and fibrinogen degradation products is restricted in healthy individuals, FDP
are over
produced in cancer patients when proteolytic enzymes, such as plasmin and
thrombin,
are released by cancer cells. In addition, FDP are produced as a by-product of
other
cancer-related processes, such as angiogenesis and metastasis. Because FDP
leak
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from tumors into surrounding fluids, elevated FDP levels can be measured in
the urine
of subjects with bladder cancer, in the plasma of lung cancer subjects, and in
the
plasma or serum of other cancer patients. The utility of FDP measurements in
cancer
diagnostics has been suspected for years; however refined assays had not been
developed that were able to quantitatively measure FDP with the sensitivity
required.
Current assays for FDP are usually restricted to measuring one specific FDP
component, such as D-dimer, as a representative of this group.
[0004] Cancer elevates fibrin and fibrinogen degradation product (FDP) levels
both
through the tissue factor (TF) and urokinase-type plasminogen activator (uPA)
regulated
pathways generating both fibrinogen degradation products and fibrin
degradation
products (FIG. 1). Cleavage of fibrinogen by plasmin produces fragments D and
E, as
the primary end-products. Thrombin converts fibrinogen to fibrin in response
to signals
from the coagulation cascade. Cleavage of fibrin by plasmin produces D-dimer
as a
primary end-product. The composition of FDP produced through cancer-induced
plasmin cleavage is influenced by the relative amounts of the two substrates;
fibrin and
fibrinogen.
SUMMARY OF THE INVENTION
[0005] Disclosed herein are methods for the detection of cancer comprising the
simultaneous detection of a defined mixture of fibrin and fibrinogen
degradation
products (FDP) containing three essential fragment types (fragment D, fragment
E, and
D-dimer) and optionally an additional three fragment types (fragment Y and two
distinguishable forms of initial plasmin digest product - IPDP) for a total of
six FDP
components. The method comprises the simultaneous detection of the three
essential
FDP antigens in a biological sample with a specific polyclonal antibody pool
in which the
polyclonal antibodies are specific for the three FDP fragments.
[0006] In one embodiment of the present disclosure, a method is provided for
detecting cancer in a subject, the method comprising the steps of: obtaining a
biological
sample from the subject; reacting the biological sample with an antibody
preparation
which binds at least three antigens associated with fibrin and fibrinogen
degradation
products (FDP) to form antibody-FDP complexes wherein the three FDP-associated
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antigens are fragment D, fragment E and D-dimer; detecting the antibody-FDP
complexes; and diagnosing cancer in the subject. In another embodiment, the
antibody
preparation additionally optionally binds to at least one of fragment Y and
initial plasmin
digest products (IPDP). In another embodiment, the antibody preparation is a
polyclonal antibody preparation.
[0007] In another embodiment, the method comprises an enzyme-linked
immunosorbent assay.
[0008] In another embodiment, the diagnosing step further comprises at least
one
additional diagnostic test.
[0009] In one embodiment of the present disclosure, a method is providing for
monitoring cancer in a patient comprising the steps of: (a) obtaining a first
biological
sample from the subject, the first sample collected at a first sampling time
point; (b)
obtaining a second biological sample from the subject, the second sample
collected
after the first sampling time point at a second sampling time point; (c)
reacting the
biological samples with an antibody preparation which binds at least three
antigens
associated with FDP to form antibody-FDP complexes wherein the three FDP-
associated antigens are fragment D, fragment E and D-dimer; (d) detecting the
antibody-FDP complexes; (e) determining the ratio of the level of FDP in the
second
biological sample to the level of FDP in the first biological sample; (f)
determining that
cancer has progressed; and optionally repeating steps (a)-(e) with additional
biological
samples taken at time points after the first and the second time points.
[0010] In another embodiment, the antibody preparation additionally optionally
binds
to at least one of fragment Y and initial plasmin digest products (IPDP). In
another
embodiment, the antibody preparation is a polyclonal antibody preparation.
[0011] In another embodiment, the method comprises an enzyme-linked
immunosorbent assay.
[0012] In another embodiment, the cancer has progressed if the ratio is
greater than
or equal to 1.15. In yet another embodiment, the cancer has regressed or is
stable if
the ratio is less than 1.15.
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[0013] In one embodiment of the present disclosure, a kit is provided for
detecting
cancer in a subject, the kit comprising an antibody preparation which binds to
at least
three antigens associated with FDP wherein the three FDP-associated antigens
are
fragment D, fragment E and D-dimer; a detection system; and instructions for
measuring the FDP and correlating the presence of the FDP with cancer.
[0014] In another embodiment, the detection system comprises a detection
antibody
specific for at least three antigens associated with FDP wherein the three FDP-
associated antigens are fragment D, fragment E and D-dimer. In another
embodiment,
the antibody preparation optionally additionally binds to at least one of
fragment Y and
initial plasmin digest products (IPDP).
[0015] In another embodiment, the antibody and detection system comprise an
enzyme-linked immunosorbent assay.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 depicts a schematic representation of cancer-induced generation
of
fibrin and fibrinogen degradation products (FDP).
[0017] FIG. 2 depicts non-reducing (FIG. 2A) and reducing (FIG. 2B) SDS-PAGE
gels of FDP immunogen lots (8 lots designated 03, 13, 15, 17, 19 21, 24 and
25). UD
refers to undigested fibrinogen; D refers to fragment D; and E refers to
fragment E.
[0018] FIG. 3 depicts a Western Blot analysis of anti-FDP antibody binding to
human serum samples (FIG. 3A, exposed to film for approximately 5 sec and FIG.
3B,
exposed to film for approximately 30 sec) from colorectal carcinoma (CRC)
patients
(n=5) and normal control serum (n=5). FIG. 3C depicts Coomassie Blue staining
of the
companion gel depicting the total amount of protein loaded per lane. The
location of
FDP subtypes are indicated on the gels.
[0019] FIG. 4 depicts immune precipitation (IP) of antigens from human serum
(normal and CRC patients) using anti-FDP polyclonal antibody pool (+) and
rabbit IgG
(-) bound beads. Three samples were tested: FDP calibrator control (FDP), a
normal
human serum (N4) and a CRC serum (C15).
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[0020] FIG. 5 depicts FDP antigens immune precipitated from the sera of five
CRC
and five normal patients using anti-FDP polyclonal antibody pool (FIG. 5A) or
rabbit IgG
(control) bound beads (FIG. 5B).
DETAILED DESCRIPTION OF THE INVENTION
[0021] Detection of fibrin and fibrinogen degradation products (FDP) can be a
valuable clinical diagnostic tool in a number of cancers. FDP levels are
correlated with
cancer occurrence, stage, progression and prognosis. Current assays for FDP
are
usually restricted to measuring one specific FDP component, such as D-dimer,
as a
representative of this group. In contrast, the present inventor has determined
that a
plurality of the fibrin and fibrinogen degradation products must be measured
simultaneously in order to correlate the presence of, or a change in the
levels of, FDP
with the presence or progression of cancer, respectively. For the purposes of
the
present disclosure, the phrase "progression of cancer" includes the
progression,
regression or re-occurrence of cancer in a patient. The presently disclosed
assay uses
an antibody preparation which specifically recognizes at least three FDP
antigens that
are particularly useful in detecting, and monitoring, the progression of
cancer. These
three essential FDP are fragment D, fragment E and the D-dimer (also known as
fragment X). Three additional FDP which are useful for detection and
monitoring cancer
are two initial plasmin digest products (IPDP), defined by sequencing, and
fragment Y.
In one embodiment of the instant method, the three essential FDP antigens are
detected approximately simultaneously in the same assay. In other embodiments,
four,
five or six FDP antigens are detected simultaneously in the same assay.
[0022] In one embodiment, the disclosed assay measures fragment D, fragment E
and D-dimer. In another embodiment, the disclosed assay measures fragment D,
fragment E, D-dimer and one or both of the IPDP. In yet another embodiment,
the
disclosed assay measures fragment D, fragment E, D-dimer and fragment Y. In
yet
another embodiment, the disclosed assay measures fragment D, fragment E, D-
dimer,
fragment Y and one or both of the IPDP.
[0023] Immunoassays are well known in the art and the affinity-purified anti-
FDP
antibodies disclosed herein can be used in different assay methods including,
but not
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limited to, enzyme-linked immunosorbent assays (ELISA), dot or slot blots,
sepharose
bead trapping analyte systems, or various rapid test formats, such as lateral-
flow or
flow-through systems.
[0024] One particular feature of the presently described method is that the
capture
and/or detection antibody is able to specifically bind to three, four, five or
six FDP
antigens. The antibody preparation is referred to herein as an antibody pool
to reflect
that the antibody preparation contains multiple antibody specificities. In one
embodiment, the antibody preparation is a polyclonal antibody preparation
raised
against an immunogen preparation which contains three, four, five or six FDP
antigen
species. Polyclonal antibodies can be generated in a variety of species
including, but
not limited to rabbit, goat, horse, donkey, sheep, chicken or shark. In
another
embodiment, antibody preparations produced against each of the three, four,
five or six
FDP antigens can be prepared independently and pooled together to form the
anti-FDP
antibody pool.
[0025] Furthermore, in other embodiments, the antibody preparation can
comprise a
pool of monoclonal, monospecific polyclonal, chimeric or humanized antibodies
or
antibody fragments in which each antibody reacts with one or more of the six
FDP
antigens as long as the antibody pool can react with three, four, five or six
FDP antigens
in the same assay approximately simultaneously. The preparation of such
antibodies is
well known in the art and will not be described in detail in this disclosure.
[0026] The present disclosure also includes systems and kits for binding to
FDP and
detecting, or monitoring, the presence or progression of cancer in a subject.
In one
embodiment, the systems comprise antibody pools and detection reagents for
binding
and detecting three, four, five or six FDP in biological samples and
correlating the
presence of the three, four, five or six FDP with the presence or progression
of cancer
in the subject.
[0027] In one embodiment, the kits comprise antibody pools and detection
reagents
for binding and detecting three, four, five or six FDP in biological samples
and for
correlating the presence of the three, four, five or six FDP with the presence
or
progression of cancer in the subject. In another embodiment, a kit can contain
one or
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more of the following in a package or container: (1) one or more antibody
pools which
bind three, four, five or six FDP to capture FDP from the biological sample;
(2) detection
antibody; (3) a solid support for immobilizing at least one of (a) the
antibody pool or (b)
the detection antibody; (4) detection reagents; (5) wash solutions; and (6)
instructions
for performing an assay using the kit components and biological samples.
Embodiments in which two or more of components (1) - (6) are found in the same
container can also be used.
[0028] When a kit is supplied, the different reagents can be packaged in
separate
containers and admixed or attached to the solid support immediately before
use. Such
packaging of the components separately can permit long-term storage without
losing
the active components' functions.
[0029] The compositions included in particular kits can be supplied in
containers of
any sort such that the life of the different components are preserved and are
not
adsorbed or altered by the materials of the container.
[0030] As stated earlier, kits can also be supplied with instructional
materials.
Instructions may be printed on paper or other substrate, and/or may be
supplied as an
electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc,
videotape, audiotape, flash memory device, etc. Detailed instructions may not
be
physically associated with the kit; instead, a user may be directed to an
internet web site
specified by the manufacturer or distributor of the kit, or supplied as
electronic mail.
[0031] Additionally the claimed methods, systems and kits can be used to
detect
cancer-associated FDP in biological samples from a variety of sources
including, but not
limited to, serum, whole blood, plasma, saliva, sputum, urine, peritoneal
fluid, tumor
tissue, cerebrospinal fluid, vaginal or rectal secretions, and tears.
[0032] When using the assay method as a screen for cancer, each laboratory
must
establish its own normal and abnormal ranges, which are based on local
population
studies. Example 11 provides a typical distribution of normal and cancer
patient
samples. Levels of FDP in biological samples from patients suspected of having
cancer
are correlated with the presence of cancer by identifying those samples that
exceed the
normal range by a predetermined number.
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[0033] When using the assay method for monitoring a patient with a confirmed
cancer diagnosis, a baseline reading should precede the evaluation of their
FDP levels.
The FDP value of the initial serum draw serves as the baseline reading. The
value of
successive serum draws are evaluated by constructing the following ratio (R);
where a =
the initial FDP value and b = the current FDP value.
R = b/a
When the ratio of the current FDP value relative to the baseline FDP value is
greater
than 1.15, the patient is likely to have disease progression, however, results
of FDP
antigen testing should be used in conjunction with other clinical modalities
that are
standard of care for monitoring disease progression in these cancer patients.
EXAMPLES
[0034] Commercially available reagents referred to in the examples were used
according to manufacturer's instructions unless otherwise indicated.
Example 1
Preparation of the FDP Immunogen
[0035] The FDP immunogen was prepared by purchasing a purified human
fibrinogen product that contained both fibrin and fibrinogen; which is
referred to as
fibrin/ogen for the purposes of this procedure. The fibrin/ogen was then
reacted with
plasmin to form fibrin and fibrinogen degradation products (FDP) by the
Haverkate and
Timan procedure (Thromb. Res. 10:803-812, 1977). The plasmin degradation of
the
fibrin/ogen was controlled by running the reaction for a specific period of
time and then
stopping the reaction with a protease inhibitor cocktail.
[0036] Specifically, the following procedure was used: Using a 50m1 conical
tube,
20m1 of MOPS buffer (5 mM 3-(N-morpholino) propane-sulfonic acid, pH 7.4
containing
0.1 M sodium chloride and 20 mM calcium chloride) was incubated in 37 C
incubator
until the solution reached 37 C temperature (approx. 20-25 min) and the
purified human
fibrin/ogen was added to the warmed MOPS buffer and agitated at 37 C until
dissolved.
One milliliter of PBS, pH 7.4 was added to plasmin to reconstitute, then 5
units of
plasmin were added to fibrin/ogen in MOPS solution. This mixture was then
agitated in
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a 37 C incubator for 3 hr. The digested FDP were removed from the shaker and
200pl
protease inhibitor cocktail was added and the solution mixed thoroughly. The
total
protein concentration of the concentrated stock FDP solution was then
measured, and
the FDP breakdown product signature was verified on a 4-20% gradient SDS-PAGE
under denaturing and reducing conditions (see FIG. 2). The resultant FDP
solution was
stored at -40 C.
[0037] The FDP immunogen was also used as in the immunoaffinity purification
of
the antibody and as an FDP calibrator and control in the FDP detection system.
Example 2
Production of Rabbit Anti-FDP Antibodies
[0038] Rabbits were immunized with FDP immunogen prepared according to
Example 1. Each rabbit received injection of an emulsion consisting of 1 mg
immunogen
in 1.0 to 1.5 ml phosphate buffer saline and equal volume of Complete Freund's
Adjuvant for the first immunization. Three weeks after the injection, each
rabbit was bled
and the serum was assayed for antibodies against FDP. One week after the
bleeding, a
booster was given to each rabbit by injection of an emulsion consisting of 1
mg
immunogen in 1 to 1.5 ml phosphate buffer saline and equal volume of
Incomplete
Freund's Adjuvant. The rabbits were maintained on a schedule of boosters and
bleeds
until they were no longer viable.
[0039] Rabbit serums obtained at various time intervals after immunization
were
assayed for the concentration of antibodies against FDP by performing a
standard titer
assay using an 96-well plate system. In a typical serum titer assay, 10 g of
immunogen
was immobilized in the wells of a 96-well microtiter plate; blocked with FBS;
then
reacted with a 1:1 dilution series of the various rabbit serums; washed;
detected with a
horseradish peroxidase (HRP) coupled goat anti-rabbit antibody (Sigma);
washed;
incubated with a 3,3',5,5'-tetramethylbenzidine (TMB) solution; stop solution,
and read
at OD 450nm. The titer was determined by graphing the OD 450nm value versus
the
dilution factor; then determining the inflection point of the curve.
[0040] The rabbit anti-FDP was immunoaffinity purified as in Example 6.
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Example 3
Production of Chicken Anti-FDP Antibodies
[0041] Chickens were immunized with FDP immunogen prepared according to
Example 1. Each chicken received injections of an emulsion consisting of 1 mg
immunogen in 1.0 to 1.5 ml phosphate buffer saline and equal volume of
Complete
Freund's Adjuvant for the first immunization (day 0). Each chicken was given
additional
immunizations on days 35, 56, 77, and 98. The chickens were bled (serum
collection)
on days 26, 40, 60, 81, and 100. Serum response testing was begun on day 45.
The
first harvest of IgY occurred on day 53. The first affinity purification began
on day 56.
[0042] Chicken serums obtained at various time intervals after immunization
were
assayed for the concentration of antibodies against FDP by performing a
standard titer
assay using an 96-well plate system. A subset of the chicken anti-FDP IgY
antibodies
were conjugated to HRP prior to the assay. In a typical serum titer assay, 10
g of
immunogen was immobilized in the wells of a 96-well microtiter plate; blocked
with FBS;
then reacted with a 1:1 dilution series of the various rabbit serums; washed;
incubated
with a TMB solution; stop solution, and read at OD 450nm. The titer was
determined by
graphing the OD 450nm value versus the dilution factor; then determining the
inflection
point of the curve.
[0043] The chicken anti-FDP was immunoaffinity purified as in Example 6.
Example 4
Production of Goat Anti-FDP Antibodies
[0044] Goats were immunized with FDP immunogen prepared according to
Example 1. Each goat received injections of an emulsion consisting of 1 mg
immunogen
in 1.0 to 1.5 ml phosphate buffer saline and equal volume of Complete Freund's
Adjuvant for the first immunization. Three weeks after the injection, each
goat was bled
and the serum was assayed for antibodies against FDP. One week after the
bleeding, a
booster was given to each goat by injection of an emulsion consisting of 1 mg
immunogen in 1 to 1.5 ml phosphate buffer saline and equal volume of
Incomplete
Freund's Adjuvant. The goats were maintained on a regime of boosters and
bleeds,
until the animal was no longer viable.
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[0045] Goat serums obtained at various time intervals after immunization were
assayed for the concentration of antibodies against FDP by performing a
standard titer
assay using an 96-well plate system. In a typical serum titer assay, 10 g of
immunogen
was immobilized in the wells of a 96-well microtiter plate; blocked with FBS;
then
reacted with a 1:1 dilution series of the various goat serums; washed;
detected with a
HRP-coupled rabbit anti-goat antibody (Sigma); washed; incubated with a TMB
solution;
stop solution, and read at OD 450nm. The titer was determined by graphing the
OD
450nm value versus the dilution factor; then determining the inflection point
of the
curve..
[0046] The goat anti-FDP was immunoaffinity purified as in Example 6.
Example 5
Preparation of FDP Coupled Sepharose CL 4B
[0047] Periodate-oxidized Sepharose CL 4B (20 ml) was washed with 10 times the
Sepharose gel volume (i.e., ten times 20 ml of Sepharose gel which equals 200
ml) of
Phosphate Buffered Saline (PBS) pH 7.5. The washed and drained Sepharose CL 4B
was mixed with 20 ml (1 gel volume) of a 2.0 mg/ml solution of the pooled FDP
antigens
(FDP in MOPS, see Example 1). Three and a half milliliters (1/6t" of the gel
volume) of
OA M sodium cyanoborohydride in deionized (DI) water was added to this
suspension
and the suspension was shaken at room temperature for 16-20 hrs. Then 40 ml (2
times the gel volume) of OA M glycine and OA M sodium cyanoborohydride in PBS
pH
7.5 was added to the suspension and the suspension was continuously agitated
for an
additional 2 hrs at room temperature. The FDP-coupled Sepharose CL-4B gel was
degassed for 15 min and then packed in a chromatographic column by gravity
flow.
The gel was washed sequentially with the following solutions: DI water (10
times the gel
volume), PBS pH 7.5 (10 times the gel volume), PBS containing 0.5M NaCl (10
times
the gel volume), OA M sodium acetate/ 0.15M NaCl pH 3 (10 times the gel
volume), PBS
pH 7.5 (10 times the gel volume). When not in use, the gel was stored in PBS
containing 0.1 % sodium azide at 4 C for up to one week.
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Example 6
Immunoaffinity Purification of Polyclonal Antibodies against FDP
[0048] Polyclonal antiserum was filtered through a 0.2 m syringe filter prior
loading
it onto the FDP-coupled Sepharose CL 4B gel column prepared as in Example 5.
The
volume of filtered polyclonal antiserum that was added is approximately equal
to the
volume of the gel (1 gel volume). After the polyclonal antiserum has been
added, the
FDP-coupled Sepharose CL 4B gel column was washed again with PBS (5 times the
gel volume) and then PBS containing 0.5M NaCl (5 times the gel volume). The
immunoaffinity purified anti-FDP antibodies that bind to the FDP on the column
were
eluted with O.1 M sodium acetate/ 0.15M NaCl pH 3. The eluent was collected in
the
fraction tubes for monitoring and later those fractions with detectable
protein
concentrations (OD 280nm greater than 0.2) were transferred to a PETG bottle
for long
term storage. The pH of the resultant antibody solution was neutralized by
adding 1M
Tris buffer pH 8.5.
Example 7
Characterization of Rabbit Polyclonal Antibodies against FDP
[0049] In one embodiment, the capture antibody is an affinity purified,
polyclonal
rabbit antibody to FDP. The rabbits were immunized and the antibodies isolated
as in
Example 3.
[0050] SDS-PAGE of the affinity purified antibodies demonstrated a high degree
of
purity. The major protein band in the non-reduced anti-FDP preparation had a
molecular
weight of approximately 152 kDa, which is consistent with the molecular weight
of rabbit
IgG. Upon reduction, the protein band of 152 kDa disappeared and,
simultaneously, two
protein bands of approximately 59 kDa and of 30 kDa appeared, which are
similar to the
molecular weights of the heavy and the light chains of an IgG molecule,
respectively.
Therefore, the anti-FDP antibodies were primarily IgG, not IgM (M.W. approx.
900 kDa),
nor IgA (a dimer of about 320 kDa). The purity of the affinity-purified FDP
antibody pool
was qualitatively estimated at about 90%, based on SDS-PAGE analysis.
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[0051] The binding of anti-FDP antibodies to FDP antigens followed a typical
Langmuir adsorption isotherm. From this data, using a double reciprocal plot,
the
binding constant of the antibodies was calculated to be 1.25x10-9 M. Because
the
antibody preparation was of polyclonal origin, the binding constant obtained
in this study
was a composite binding constant, i.e. an average of binding constant
contributed by
antibodies produced by different clones.
[0052] The capture and detection antibodies ensure the specificity of any
ELISA
based test. The FDP antibody pool was cross-reacted with human serum samples
in
both Western Blots (Fig. 3) and in immune precipitation (IP) experiments
(FIGs. 4 and 5
and Table 2) in order to verify that the FDP antibody pool captures FDP in
human
serum samples. In addition, the proteins that were immunoprecipitated by the
FDP
antibody pool were sequenced and identified using mass spectrometry.
[0053] Western blot analysis of human serum samples from both colorectal
cancer
(CRC) and normal control patients were used to determine the number and the
estimated molecular weights of the FDP antigens in human serum. FIGs. 3A and
3B
depict Western blots of the same gel exposed to film for 5 sec and 30 sec
respectively.
FIG. 3C depicts the corresponding Coomassie-stained gel. In FIG. 3, six major
FDP
antigens were detected in CRC serum samples with estimated molecular weights
of
340, 260-320, 220, 150, 80 and 50 kDa. These bands correspond with undigested
fibrinogen, initial plasmin digest products (IPDP), fragment D-dimer/fragment
X,
fragment Y, fragment D and fragment E, respectively. In contrast, only one
major band,
at approximately 220 kDa, was detected in normal control sera. For all of the
serum
samples tested, the intensity of the reactions was much higher in the serum
samples
from CRC patients than from the normal controls.
Table 1. Analysis of anti- FDP cross reactivity in cancer vs. normal serum
Average of the Mean
Estimated Ratio of Averaged
Fibrinogen MW Area Densities from Mean Densities p-value
Degradation Individual Subjects
Products
(kDa) (in2) Cancer Normal Cancer/Normal
n=5 n=5
Undigested 340 0.1 50.3 4.0 12.6 5.1E-08
& IPDP
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IPDP 260-320 0.4 150.7 4.1 36.7 1.3E-10
Fragment X/
Fragment D- 220 0.2 131.1 14.8 8.8 4.0E-10
dimer
Fragment Y 150 0.2 77.4 5.1 15.2 5.4E-10
Fragment D 80 0.2 51.0 3.8 13.5 5.4E-09
monomer
Fragment E 50 0.2 39.1 3.3 11.9 5.0E-08
monomer
[0054] As demonstrated in Table 1, the average signal densities of all six of
these
FDP components were significantly (p-values < 0.00000005) higher in the serum
samples from the five CRC patients than from the five normal control patients.
Densitometry-based analysis of cross-reactivity levels in the Western blot
(FIG. 3A)
used Scion Image densitometry software (Scion Corporation). Table 1 confirms
that
anti-FDP pool can distinguish between cancer and normal serum samples, based
on
their respective FDP levels.
[0055] In order to verify the number and sizes of the FDP antigens captured
from
human serum samples, immune precipitation studies were performed using either
rabbit
anti-FDP IgG or control rabbit IgG bound to sepaharose beads (FIGs. 4 and 5).
Immune
precipitation also concentrated the FDP antigens such that they could be
sequenced
from a Coomassie stained SDS-PAGE.
[0056] Using the Affi-Gel Hz Immunoaffinity Kit (BIO-RAD) according to the
manufacturer's instructions, the rabbit anti-FDP IgG or the control rabbit IgG
antibodies
were covalently linked to Sepharose beads via a conserved carbohydrate moiety
in the
Fc portion on the IgG heavy chain. Human serum samples were selected at random
and diluted 1 to 10 in PBS, pH 7.4. The diluted serum samples were incubated
with
either the anti-FDP-bound beads or control rabbit IgG-bound beads. In
addition, both
types of beads were incubated with a 0.25 mg/ml solution of prepared FDP to
serve as
a positive control for the IP reactions. The IP reactions between the diluted
sera and the
control rabbit IgG bound beads serve as a control for recognizing false
positive
reactions between the non-specific rabbit IgGs and serum antigens. The IP
reactions
were incubated overnight at 4 C on an end-over-end rotator. The IP beads were
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washed five times with PBS, and the antigens were extracted in non-reducing
Laemmli
sample buffer at 95 C for 8 minutes. Then the extracted antigens from the anti-
FDP and
control rabbit IgG beads were run on a 4-20% SDS-PAGE. FIG. 4 shows a complete
set of experimental conditions for two representative serum samples; N4 is
from normal
patient 4 and C15 is from colorectal cancer patient 15. FIGs. 5A and 5B reveal
the
reproducibility of these IP reactions by showing five additional normal and
five additional
colorectal cancer patients in the IP assay.
[0057] The results of the IP experiment, presented in FIG. 4, reveal that four
major
FDP-specific antigens and one non-specific antigen have been captured from the
serum
of the colorectal cancer patient serum by the anti-FDP beads (+). From this
sample
(C15(+), 2nd to last lane from the right), the estimated molecular weights
(MW) of the
four major FDP-specific antigens are 340, 300, 220, and 50 kDa, respectively,
in
descending order. The one non-specific antigen in this lane is approximately
25 kDa.
The control lanes N4(-) and C15(-) (3rd and 5th lanes from the right) for the
serum
samples also show a reaction with a 25 kDa protein, which corresponds with the
25 kDa
protein in the N4 and C15 samples exposed to anti-FDP beads (+), as shown in
N4(+)
and C15(+) (2nd and 4th lanes from the right). The presence of the 25 kDa
protein in all
of these lanes, N4(-), N4(+), C15(-), C15(+), confirms that the 25 kDa protein
extracted
from the IP reactions of serum samples non-specifically interacts with any IgG
bound
bead. The positive control reactions for the IP assay are designated FDP(-)
and FDP(+)
(6th and 7 th lanes from the left). The bead control lane (FDP(-)) reveals no
interactions
between the FDP calibrator solution, which is not serum based, and the
negative control
beads. The FDP(+) reaction contains one minor band and three major bands. The
estimated MW of the one minor band is 340 kDa, which corresponds with the MW
of
undigested fibrinogen. The estimated molecular weights of the three major FDP-
specific
antigens in the FDP control immunogen solution are 220 kDa (which corresponds
with
the MW of the fibrinogen fragment X or D-dimer), 80 kDa (which corresponds
with the
MW of fibrinogen fragment D monomer) and 50 kDa (which corresponds with the
fibrinogen fragment E monomer). The anti-FDP beads captured four major FDP
antigens from the human serum of a colorectal cancer patient; whereas, the
same
beads only brought one minor (faint band) in the normal serum at approximately
53kDa.
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Comparisons between the IP reactions of the human serum samples (and FDP) with
the
anti-FDP beads versus the control beads confirms the specificity of the anti-
FDP
antibody pool.
[0058] FIG. 5 reveals that the pattern of FDP antigens is reproducible among
multiple patient samples. For this experiment, sera from an additional five
CRC and five
normal patients (chosen at random from clinical trial samples) were tested in
immune
precipitation reactions, separately, with both the control rabbit IgG and the
anti-FDP
beads. In addition, the FDP immunogen (calibrator solution) was tested with
both types
of beads. The SDS-PAGE in FIG. 5A contains the extracted FDP-specific antigens
from
the FDP (positive control), the CRC patient serum samples, and the normal
serum
samples. The SDS-PAGE shown in FIG.5B shows the antigens adhering to the
control
rabbit IgG beads from the same samples and in the same order as in FIG. 5A.
[0059] The experiment shown in FIG. 5 confirms that there are four major FDP-
specific antigens in human sera, and one non-specific antigen at 25 kDa. In
FIG. 5A,
the FDP control lane, which is derived from an in vitro plasmin digestion of
fibrin and
fibrinogen under controlled conditions, contains three major bands and one
minor band.
Based on the approximate molecular weights of these non-reduced samples, the
major
bands captured by the FDP antibody pool from the FDP control sample correspond
to
fibrinogen fragment D-dimer, fragment D monomer, and fragment E monomer, in
descending order relative to the molecular weights. The minor band at 340 kDa
is
presumably undigested fibrinogen, also based on its approximate molecular
weight. The
FDP-specific antigens from human sera are at approximately 340, 300 (280-320),
210,
and 50 kDa. These bands correspond with undigested fibrinogen, initial plasmin
digest
products (IPDP), fragment D-dimer/fragment X, and fragment E, respectively.
The IP
reactions in this (and all of the other IP experiments) were normalized by
adding
equivalent concentrations of each serum sample to the IP reactions. In FIG.
5B, the
predominant antigen is at approximately 25 kDa. As discussed previously, this
indicates
that the serum protein at 25 kDa reacts non-specifically with all rabbit IgG
bound beads.
The 25 kDa protein also apparently interacts non-specifically with the rabbit
anti-FDP
IgG bound bead reactions shown in FIG. 5A.
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Table 2. Quantification of DR-70 (FDP)-specific antigens from CRC patient
sera
Average of the Mean
Estimated Ratio of Averaged
Fibrinogen MW Area Densities from Mean Densities p-value
Degradation Individual Subjects
Products
(kDa) (in 2) Cancer Normal Cancer/Normal
n=5 n=5
Undigested 340 0.1 42.5 7.1 6.0 1.5E-06
fibrinogen
I P D P 280-320 0.3 61.8 7.8 7.9 4.2E-08
Fragment X
& Fragment 210 0.3 57.0 8.7 6.6 3.9E-09
D-dimer
Fragment E 50 0.3 69.0 9.2 7.5 3.5E-05
Non-specific 25 0.3 64.1 68.1 0.9 5.3E-01
[0060] In this experiment shown in FIG. 5, the FDP specific antigens from the
CRC
patient sera were between 6.6 and 9.8 times more abundant than that from the
normal
patient sera, based on densitometry readings presented in Table 2. However,
the non-
specific antigen (approximately 25 kDa) had a ratio of cancer relative to non-
cancer that
was equal to 0.9 or approximately 1. As demonstrated in Table 2, the average
signal
densities of all of the FDP-specific antigens were significantly (p-values <
0.000035)
higher in the serum samples from the five colorectal cancer patients than from
the five
normal control patients. The non-specific antigen band from the sera of cancer
patients
was not significantly (p = 0.53) different from that of normal controls. Table
2 confirms
that anti-FDP antibody pool can distinguish between cancer and normal serum
samples
based on their respective FDP levels.
Example 8
Characterization of the Anti-FDP Capture Antibody
[0061] The capture antibody pool was produced and characterized in one
embodiment as described in Examples 2 and 7.
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Example 9
FDP Antibody Conjugated to Horseradish Peroxidase (HRP) for Detection
[0062] The detection antibody was enzyme labeled using horseradish peroxidase
(HRP) for detection in the assay. More specifically, in this example the
detection
antibody was a polyclonal rabbit anti-human FDP antibody produced according to
the
methods of Example 2. The antibody is the purified immunoglobulin fraction of
rabbit
antiserum conjugated with horseradish peroxidase of very high specific
enzymatic
activity.
[0063] The detection antibody immunogen included both fibrin and fibrinogen
degradation products. The antibody reacts with native human fibrinogen as well
as with
the FDP fragment subtypes: D, E, X/D-dimer, and Y. Traces of contaminating
antibodies
were removed by solid-phase absorption with human plasma proteins. The
specificity
of the antibody was ascertained by Western blot analysis. The detection
antibody has
confirmed cross-reactivity with the same six FDP fragments in human serum of
cancer
patients as was detected by the capture antibody.
Example 10
Enzyme Linked Immunosorbant Assay (ELISA) for FDP
[0064] The FDP ELISA is an enzyme-labeled, sandwich immunoassay. The
capture antibodies (polyclonal, rabbit anti-FDP) were derived from sera of
rabbits as
described in Examples 2, 7 and 8.
[0065] In an exemplary embodiment, the FDP immunoassay involves the use of
removable strips in a 96-well, microtiter plate format. The wells of the
microtiter plate
were coated with affinity purified rabbit anti-FDP antibodies. Human serum
samples
were diluted (1:200) and were applied to the wells. FDP was captured from the
serum
samples by the anti-FDP antibodies immobilized on the well of the microtiter
plate. After
a wash step, anti-FDP antibodies conjugated to HRP (detection antibody) were
added
to the wells. The anti-FDP-HRP complex binds to the captured FDP to form an
immunological sandwich with the immobilized anti-FDP antibodies. After a
second
wash step, the enzyme substrate TMB was added to the well. The end point was
read
in a micro plate reader at 450 nm after the reaction was stopped with 0.1 N
HCI. The
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intensity of the color formed is proportional to the amount of FDP in the
serum. The
amount was quantified by interpolation from a standard curve using purified
FDP
calibrators.
Example 11
Distribution of FDP in Serum from Normal Healthy Individuals and in Patients
with
Benign Disease
[0066] To determine the approximate distribution of FDP values in a normal
healthy
cohort, a sample of 209 women and 212 men who thought themselves to be disease
free on the day of the draw was utilized. Subjects were divided by sex and by
age with
the two age groups: 40-64 years and 65 years plus. Values for the assay were
determined in duplicate. A cumulative distribution was established. Order
statistics for
every 5th percentile were determined.
[0067] A variety of samples from subjects with benign disease were assembled
to
determine the distribution of serum FDP values in benign diseases that may be
co-
existent in patients with confirmed cancer. Specimens were collected under IRB
approved protocols with the appropriate informed consent from geographically
diversified sites across the United States. Table 3, below, describes the
numbers of
samples and the makeup of the benign disease groups. A total of 400 patients
were
tested in this cohort including 74 normal samples.
Table 3. Distribution of Diseases
Benign Condition Number
Benign Gastrointestinal GI Disease 61
Benign Genitourinary (GU) Disease 94
Heart Disease/HTN 87
Normal 74
Benign Pancreatic Disease 84
Grand Total 400
[0068] Table 4 shows the characteristics of each of the benign groups tested
as well
as those for normal individuals. There is significant scatter within all the
groups with the
exception of the normal cohort. Mean values tend to be skewed by samples with
high
FDP values. However, the median value in each and every one of these groups is
well
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within the reference interval for apparently healthy individuals. As in all
other studies in
this trial, FDP values were determined in duplicate with all subjects
consented under an
IRB approved protocol.
[0069] Analysis shows that the median values of these benign disease cohorts
are
of the same magnitude as the median value of the normal healthy subjects. All
benign
conditions except for those of the pancreas have distributions and 95%
confidence
limits similar to the normal healthy cohort.
Table 4. Sample Statistics by Benign Disease State
Disease State FDP
Normal N 74
Mean .81
Median .77
Std. Deviation .284
Minimum .18
Maximum 1.80
Heart Disease N 87
Mean 163.6
Median 1.0
Std. Deviation 337.53
Minimum .14
Maximum 1864.8
Benign Pancreatic N 84
Disease
Mean 262.8
Median 1.2
Std. Deviation 529.00
Minimum .29
Maximum 1902
Benign GI Disease N 61
Mean 29.3
Median .798
Std. Deviation 138.59
Minimum .30
Maximum 924.3
Benign GU Disease N 94
Mean 10.9
Median .63
Std. Deviation 98.89
Minimum .17
Maximum 959.57
Total N 400
Mean 97.9
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Median .81
Std. Deviation 314.4
Minimum .14
Maximum 1901.
Example 12
Distribution of FDP in Serum from Subjects with Malignant Disease
[0070] This study was conducted in 439 patients who had been diagnosed with,
and
were being treated for, a primary malignancy. The subjects were categorized
into five
groups: lung/liver cancer, breast/ovarian/cervical cancer, gall
bladder/biliary/pancreatic
cancer, gastric cancer and colorectal cancer. The FDP assay was performed in
duplicate on the samples. A one-way ANOVA was performed with post hoc Tamhane
tests. The results indicate that there are no significant differences between
the groups.
The details are shown in Table 5 for each cancer group.
Table 5. Sample Statistics by Cancer Group
Cancer Group FDP
N 89
Mean 4.05
Lung/Liver Cancer Median 1.65
Std. Error of Mean 1.10
Minimum .17
Maximum 76.12
N 90
Mean 3.43
Breast/Cervical/Ovarian Cancer Median 1.67
Std. Error of Mean .53
Minimum .31
Maximum 39.23
N 47
Mean 3.54
Gallbladder/Pancreas Cancer Median 2.29
Std. Error of Mean .75
Minimum .64
Maximum 34.00
Gastric/Other Cancers N 26
Mean 34.99
Median 2.24
Std. Error of Mean 32.22
Minimum .67
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Maximum 840.54
N 187
Mean 3.43
Colorectal Cancer Median 1.33
Std. Error of Mean 1.38
Minimum .24
Maximum 259.28
N 439
Mean 5.44
Total Cancers Median 1.61
Std. Error of Mean 2.01
Minimum .17
Maximum 840.54
Example 13
Distribution in percent of FDP values within each disease cohort
[0071] In each normal, benign and disease cohort above, the values for FDP
were
analyzed for the different FDP concentration levels within each disease
cohort. The
StatXact software was utilized during this analysis to establish the exact
95%
confidence intervals for the statistics. The distribution table is presented
in Table 6.
Table 6. Distribution of percent of FDP values
Percent (%)
95% Cl (lower%, upper
Disease s u b je c t s 0-1.4 pg/ml 1.5-2.4 pg/ml 2.5-4.9pg/ml > 5.0 pg/ml # of
Normal 420 94.5 5.0 0.5 0.0
(91.9, 96.3.1,7.5 (0.1, 1Ø0,0.9
< 65 years 337 96.4 3.3 0.3 0.0
(93.9, 98.2) 1.6, 5.8 (0.0, 1.6) (0.0, 1.1)
> 65 years 83 86.8 12.1 1.2 0.0
(77.5, 93.2) 5.9, 21.0 0.0, 6.5) 0.0, 4.4
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Benign 326 75.5 6.8 0.6 17.2
(70.4, 80.0) (4.3, 10.0) 0.1, 2.2 (13.2, 21.7)
GU Disease 94 94.7 4.3 0.0 1.1
(88.0, 98.3) (1.2, 10.5) 0.0, 3.9 0.0, 5.8
GI Disease 61 90.2 3.3 0.0 6.6
(79.8, 96.3) (0.4, 11.4) 0.0, 5.9 (1.8, 16.0)
Pancreas 84 60.7 15.5 2.4 21.4
(49.5, 71.2) 8.5, 25.0 0.3, 8.3 (13.2, 31.7)
Heart Disease 87 58.6 3.5 0.0 37.9
(47.6, 69.1) 0.7, 9.8 0.0, 4.2 (27.7, 49.0)
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Malignant 439 44.0 24.2 19.6 12.3
(39.3, 48.8) (20.2, 28.4) (16.0, 23.6) (9.4, 15.7)
Colon 187 55.6 21.4 (15.7, 15.0 (10.2, 8.0 (4.6,
(48.2, 62.9) 28.0) 20.9) 12.9)
Lung 44 34.1 38.6 18.2 9.1
(20.5, 49.9) (24.4, 54.5) (8.2,3 .7 2.5, 21.7
Liver 44 31.8 27.3 22.7 18.2
(18.6, 47.6) (15.0, 42.8) (11.5, 37.8) 8.2, 32.7
Breast 31 54.8 25.8 12.9 6.5
(36.0, 72.7) (11.9, 44.6) 3.6, 29.8 0.8, 21.4
Ovarian 31 25.8 6.5 32.3 35.5
(11.9, 44.6) 0.8, 21.4 (16.7, 51.4) (19.2, 54.6)
Cervical 28 50.0 28.6 7.1 14.3
(30.7, 69.4) (13.2, 48.7) 0.9, 23.5 4.0, 32.7
Gall Bladder 19 42.1 26.3 31.6 0.0
(20.3, 66.5) 9.2, 51.2 12.6, 56.6 (0.0, 17.7)
Pancreas 28 25.0 17.9 35.7 21.4
(10.7, 44.9) 6.1, 36.9 (18.6, 55.9) 8.3, 41.0
Gastric/ Other 27 22.2 33.3 29.6 14.8
8.6, 42.3 (16.5, 54.0) (13.8, 50.2) 4.2, 33.7
Exact binomial confidence limits.
Example 14
Serial (longitudinal) monitoring of patients diagnosed with colorectal cancer
[0072] Samples for this portion of the study were obtained from two
retrospective
sample banks. Forty-eight serial sets were obtained from Geffen Cancer Center
in Vero
Beach, FL and sixty-four serial sets were taken from the serum banks at MD
Anderson
Cancer Center in Houston, TX.
[0073] Out of a total of 445 evaluable observations, there were 112 evaluable
patient serial sets. The samples for the serial monitoring study were
retrospective
banked samples that were collected blindly and without bias to include all
patients with
diagnosed colorectal cancer in the bank at the time of the collection. The
average
number of observations per patient is 4Ø
[0074] In summary, serial samples were taken from 112 colon cancer patients
resulting in a total of 445 paired observations in which a FDP reading and a
determination of disease progression were obtained. The sequential draws
covered an
average longitudinal period of at least nine months. Progression of the FDP
value in the
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serial monitoring set was evaluated as a percentage change between the current
and
previous readings (Y) and 15% was determined to be the minimum percentage to
specify disease progression in the FDP assay. Clinical disease progression (D)
was
determined by the subject's physician based on those office procedures and
clinical
laboratory based analyses that were the standard of care during the time of
the
monitoring period. The primary objective of this analysis is to demonstrate
that the FDP
assay was informative by showing that the sum of sensitivity and specificity
exceeds 1.
[0075] The response to therapy was evaluated using information provided in the
records by the clinicians based on the results of clinical examinations and
imaging
results (i.e. bone scans, CT scans, magnetic resonance imaging studies,
radiography,
or ultrasound). Response to therapy is defined as follows:
[0076] Complete response or no evidence of disease (CR or NED): The complete
disappearance of all clinical and image-measurable disease as evidenced by the
clinical
exam and imaging or other diagnostic modalities as ordered by the physician.
[0077] Partial Response (PR): In patients with metastases at the time of the
original
draw, a noticeable reduction in the size of primary metastatic lesions or bone
metastases demonstrating at least stabilization as observed on the bone scan.
[0078] Stable Disease (SD): No significant change in the size of primary
metastatic
lesions or no noticeable increase in the size of primary lesions or no new
lesions as
evidenced by the clinical exam and imaging or other diagnostic modalities as
ordered by
the physician.
[0079] Progressive Disease (PD): Clinical or imaging results that clearly
indicate the
presence of lesions not seen on previous examinations or a significant
increase in the
size of primary or metastatic lesions.
[0080] Serial samples were taken from 112 colon cancer patients resulting in
446
paired observations in which the FDP assay reading and a determination of
disease
progression were obtained. Since several patients had signs of progression
even at the
first examination, it was decided to determine the relationship between FDP
assay value
and progression at successive visits. Thus, from the data, a variable was
derived by
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taking the ratio of a subsequent FDP assay reading and the previous reading.
This
measure was intended to determine the increase from a previous reading as a
means of
providing information on progression. A determination was made that a
meaningful
increase to determine evidence of progression was 15% increase or more. Thus,
if the
ratio was 1.15 or higher, the FDP assay value was deemed to be positive,
otherwise it
was deemed to be negative and this determination was paired with the finding
at that
visit of progression or not.
[0081] The resulting 335 paired observations from the post baseline sampling
were
evaluated in two ways, per-visit and per-patient evaluations. The initial
analysis
performed a bootstrap sample for each patient by randomly sampling one visit
at for
each sample and recording the sensitivity or specificity for that visit. Note
that if there
was a progression and the sensitivity would be 1 if the FDP assay value
increased from
the previous visit by 15% or more and 0 if it did not. If there were no
progression at that
visit, then there would be no sensitivity reported at that visit, but the
specificity would be
reported as a 1 if the FDP assay value was below a 15% increase for that visit
and 0
otherwise. For the per-visit analysis, there were 135 visits for sensitivity
and 198 visits
for specificity.
[0082] A second analysis was done on a per-patient basis in which the number
of
progressions across all visits for a given patient was used to compute a
patient level
sensitivity by taking the number visits that FDP assay values increase by at
least 15%
among the number of visits that there was a progression. Similarly, the number
of visits
at which FDP assay values had a lower than 15% increase divided by the number
of
visits in which there was a non-progression allowed the computation of a per-
patient
specificity. Recall that if a patient had all progressions there would be no
specificity for
that patient and if a patient had all non-progressions, there would be no
sensitivity for
that patient. This resulted in a sample of 112 patients with at least one
sensitivity,
specificity, or both. This resulted in 70 estimates of per-patient sensitivity
and 86
estimates of per-patient specificity.
[0083] Positive (PPV) and negative (PNV) predictive values were also computed
on
a per-patient and per-visit basis. The PPV is calculated by dividing the
number of times
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progression was present when the FDP assay value increased by 15% or more and
NPV by dividing the number of times progression was absent when FDP assay
values
did not increase by 15% or more.
[0084] When small samples are available for evaluation and may have a complex
structure, a statistical method called the bootstrap may be used to provide
sound
estimates of relevant population properties. A method to allow such a
computation is to
take repeated random samples with replacement from the 333 observations and
compute the sum of the sensitivity and specificity and then determine through
the
frequency distribution of the resulting samples the sum that excludes the
percentile
corresponding to the alpha level of the statistical test. Such samples
automatically
incorporate any intra-patient correlations and yield an unbiased test of the
null
hypothesis above. Bootstrap sampling is usually repeated a number of times to
be
certain that the resulting distributions are consistent from run to run.
[0085] The per-visit bootstrap sample was done 2,000 times by randomly
sampling
one visit for each of the 112 patients with replacement. From each sample, the
mean
sensitivity, means specificity, and the sum were computed by summing the
sensitivity or
specificity (the 1 or 0 values) over all patients and dividing by the total
number of
sensitivity or specificity measurements present. After the mean sensitivity
and
specificity for a sample was obtained, the two were added together to obtain
the sum.
This exercise resulted in 2000 bootstrap estimates of the per-visit
sensitivity, specificity,
and the sum of the two. A frequency display of these 2000 values by variable
allows
the boot strap estimation of the lower one- and two-sided 95% confidence
limits as well
as other.
[0086] The method of sampling was done by assigning each of the k (between 1
and 7) visits per-patient a number between 1 and k. A sample of 112
observations was
obtained by generating a uniform random number between 0 and 1, multiplying
that
number by k, taking the integer of that number, and adding 1. This process
provided a
random number between 1 and k for each patient. The FDP assay ratio of change
from
previous was captured for the patient visit with the number obtained.
Duplicates were
allowed because sampling was done with replacement and the process was
repeated
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112 times. The sensitivity, specificity, and the sum were computed for each
sample of
112 and retained.
[0087] The computed per-visit sensitivity from the 333 per-visit evaluations
was
10088/134= 65.19, the specificity was 100133/199= 67.34, the sum of
sensitivity and
specificity was 132.53, the PPV was 10088/153= 57.52, and the NPV was
100134/181= 74.03.
[0088] The results of the statistical analysis to determine the effectiveness
of the
FDP immunoassay for providing information on disease progression in colon
cancer
patients are provided in Table 7 below.
Table 7. Results of Five Repetitions of 2,000 Samples of 112 per-visit
Observations of the Sensitivity, Specificity, the Sum of Sensitivity and
Specificity,
PPV, and NPV
Run Measure Median Lower 5% Lower 2.5%
1 Sensitivity 65.85 55.81 54.17
Specificity 66.20 59.09 57.63
Sensitivity + Specificity 132.66 120.31 118.23
PPV 58.33 50.91 49.23
NPV 73.13 66.18 65.45
2 Sensitivity 65.85 56.25 54.17
Specificity 67.19 58.46 56.92
Sensitivity + Specificity 132.92 120.75 118.72
PPV 58.82 50.91 48.98
NPV 73.21 66.18 64.91
3 Sensitivity 65.91 56.10 54.00
Specificity 67.16 58.73 56.92
Sensitivity + Specificity 133.20 120.29 117.81
PPV 58.93 49.15 48.94
NPV 73.33 66.18 65.08
4 Sensitivity 65.85 56.25 53.66
Specificity 66.67 58.57 56.72
Sensitivity + Specificity 132.96 119.73 116.99
PPV 58.73 49.18 48.84
NPV 73.13 66.00 64.52
Sensitivity 65.96 56.25 54.90
Specificity 67.16 58.73 57.38
Sensitivity + Specificity 133.29 120.62 118.23
PPV 59.02 50.88 49.09
NPV 73.24 66.20 64.91
[0089] Whether the test is done at the alpha = 0.05 or at the alpha = 0.025,
the sum
of sensitivity and specificity from this analysis clearly is statistically
significantly above
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100. The median sum is likely to be about 133 with the median sensitivity
about 65 and
the median specificity about 67. The lower one-sided 5% confidence bound for
the
sum is about 120 and the lower one-sided 2.5% confidence bound is about 118.
The
median PPV and NPV are about 59 and 73, respectively, across the five samples.
Note
that these values are consistent with those computed from the per-visit values
given
above in Table 7. The five repetitions of the sample of 2,000 demonstrate that
the
result is robust and consistent.
[0090] For the per-patient analysis, the computed per-visit sensitivity from
the 112
per-patient evaluations was 10045.68/69 = 66.21, the specificity was
10058.63/86=
68.18, the sum of sensitivity and specificity was 134.39, the PPV was
10051.83/97=
53.44, and the NPV was 10071.67/103= 69.58.
Table 8
Run Measure Median Lower 5% Lower 2.5%
1 Sensitivity 66.12 58.78 57.11
Specificity 68.20 62.58 61.46
Sensitivity + Specificity 134.38 123.82 121.46
PPV 53.30 45.88 44.55
NPV 69.74 62.71 60.90
2 Sensitivity 66.17 58.58 57.13
Specificity 68.16 62.39 61.41
Sensitivity + Specificity 134.26 123.85 122.17
PPV 53.41 45.34 43.96
NPV 69.50 62.75 61.37
3 Sensitivity 66.13 58.59 57.27
Specificity 68.20 62.29 61.23
Sensitivity + Specificity 134.32 123.79 121.36
PPV 53.37 45.71 43.89
NPV 69.50 62.06 60.89
4 Sensitivity 66.43 58.53 56.99
Specificity 68.28 62.64 61.55
Sensitivity + Specificity 134.69 123.87 121.17
PPV 53.70 45.92 44.39
NPV 69.55 62.71 61.33
Sensitivity 66.42 59.00 57.92
Specificity 68.29 62.82 61.64
Sensitivity + Specificity 134.62 124.38 122.92
PPV 53.41 45.75 44.15
NPV 69.64 62.66 61.38
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[0091] Whether the test is done at the alpha = 0.05 or at the alpha = 0.025,
the sum
of sensitivity and specificity from this analysis clearly is statistically
significantly above
100. The median sum is likely to be about 134 with the median sensitivity
about 66 and
the median specificity about 68. The lower one-sided 5% confidence bound for
the
sum is about 124 and the lower one-sided 2.5% confidence bound is about 121.
The
median PPV and NPV are about 53 and 69, respectively, across the five samples.
Note
that these values are consistent with those computed from the per-patient
values given
above Table 8. The five repetitions of the sample of 2,000 demonstrate that
the result is
robust and consistent.
[0092] The conclusion from the analysis of the effectiveness of the FDP
immunoassay for monitoring colorectal cancer samples is as follows. These data
and
analyses demonstrate that the FDP assay, when taken as a 15% or greater change
from the previous visit, yields informative data regarding colon cancer
progression. The
FDP immunoassay results must be used in conjunction with standard of care
procedures for monitoring colorectal cancer patients.
[0093] Unless otherwise indicated, all numbers expressing quantities of
ingredients,
properties such as molecular weight, reaction conditions, and so forth used in
the
specification and claims are to be understood as being modified in all
instances by the
term "about." Accordingly, unless indicated to the contrary, the numerical
parameters
set forth in the specification and attached claims are approximations that may
vary
depending upon the desired properties sought to be obtained by the present
invention.
At the very least, and not as an attempt to limit the application of the
doctrine of
equivalents to the scope of the claims, each numerical parameter should at
least be
construed in light of the number of reported significant digits and by
applying ordinary
rounding techniques. Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations, the
numerical values
set forth in the specific examples are reported as precisely as possible. Any
numerical
value, however, inherently contains certain errors necessarily resulting from
the
standard deviation found in their respective testing measurements.
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[0094] The terms "a," "an," "the" and similar referents used in the context of
describing the invention (especially in the context of the following claims)
are to be
construed to cover both the singular and the plural, unless otherwise
indicated herein or
clearly contradicted by context. Recitation of ranges of values herein is
merely intended
to serve as a shorthand method of referring individually to each separate
value falling
within the range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually recited herein.
All methods
described herein can be performed in any suitable order unless otherwise
indicated
herein or otherwise clearly contradicted by context. The use of any and all
examples, or
exemplary language (e.g., "such as") provided herein is intended merely to
better
illuminate the invention and does not pose a limitation on the scope of the
invention
otherwise claimed. No language in the specification should be construed as
indicating
any non-claimed element essential to the practice of the invention.
[0095] Groupings of alternative elements or embodiments of the invention
disclosed
herein are not to be construed as limitations. Each group member may be
referred to
and claimed individually or in any combination with other members of the group
or other
elements found herein. It is anticipated that one or more members of a group
may be
included in, or deleted from, a group for reasons of convenience and/or
patentability.
When any such inclusion or deletion occurs, the specification is deemed to
contain the
group as modified thus fulfilling the written description of all Markush
groups used in the
appended claims.
[0096] Certain embodiments of this invention are described herein, including
the
best mode known to the inventors for carrying out the invention. Of course,
variations
on these described embodiments will become apparent to those of ordinary skill
in the
art upon reading the foregoing description. The inventor expects skilled
artisans to
employ such variations as appropriate, and the inventors intend for the
invention to be
practiced otherwise than specifically described herein. Accordingly, this
invention
includes all modifications and equivalents of the subject matter recited in
the claims
appended hereto as permitted by applicable law. Moreover, any combination of
the
above-described elements in all possible variations thereof is encompassed by
the
invention unless otherwise indicated herein or otherwise clearly contradicted
by context.
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[0097] Specific embodiments disclosed herein may be further limited in the
claims
using consisting of or consisting essentially of language. When used in the
claims,
whether as filed or added per amendment, the transition term "consisting of
excludes
any element, step, or ingredient not specified in the claims. The transition
term
"consisting essentially of limits the scope of a claim to the specified
materials or steps
and those that do not materially affect the basic and novel characteristic(s).
Embodiments of the invention so claimed are inherently or expressly described
and
enabled herein.
[0098] Furthermore, numerous references have been made to patents and printed
publications throughout this specification. Each of the above-cited references
and
printed publications are individually incorporated herein by reference in
their entirety.
[0099] In closing, it is to be understood that the embodiments of the
invention
disclosed herein are illustrative of the principles of the present invention.
Other
modifications that may be employed are within the scope of the invention.
Thus, by way
of example, but not of limitation, alternative configurations of the present
invention may
be utilized in accordance with the teachings herein. Accordingly, the present
invention
is not limited to that precisely as shown and described.
31
