U.S. patent application number 15/435880 was filed with the patent office on 2017-06-08 for assay to measure midkine or pleiotrophin level for diagnosing a growth.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Jeffrey Baron, Youn Hee Jee.
Application Number | 20170160279 15/435880 |
Document ID | / |
Family ID | 49679676 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170160279 |
Kind Code |
A1 |
Baron; Jeffrey ; et
al. |
June 8, 2017 |
ASSAY TO MEASURE MIDKINE OR PLEIOTROPHIN LEVEL FOR DIAGNOSING A
GROWTH
Abstract
The invention provides methods and kits for diagnosing a growth
in a subject by providing a sample of a growth taken from a
subject, determining the level of midkine or pleiotrophin in the
sample by an immunoassay, and comparing the level of midkine or
pleiotrophin determined from the sample with a control. An
increased level of midkine or pleiotrophin in the sample as
compared to the control is diagnostic of a malignant growth,
whereas an equivalent or decreased level of midkine or pleiotrophin
in the sample as compared to the control is diagnostic of a benign
growth.
Inventors: |
Baron; Jeffrey; (North
Potomac, MD) ; Jee; Youn Hee; (Bethesda, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
49679676 |
Appl. No.: |
15/435880 |
Filed: |
February 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14646078 |
May 20, 2015 |
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PCT/US13/70305 |
Nov 15, 2013 |
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15435880 |
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61815342 |
Apr 24, 2013 |
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61728624 |
Nov 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/475 20130101;
G01N 2800/00 20130101; G01N 33/543 20130101; G01N 33/57488
20130101; G01N 33/53 20130101; G01N 33/57407 20130101; A61B 10/02
20130101; G01N 2333/47 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; A61B 10/02 20060101 A61B010/02 |
Claims
1-38. (canceled)
39. A method of treating a growth in a subject comprising: (a)
providing a sample of a growth obtained from thyroid tissue of a
subject, (b) analyzing the sample by an immunoassay to determine
the concentration of midkine protein in the sample, (c) analyzing
the sample by an immunoassay to determine the concentration of
thymoglobulin protein in the sample, (d) determining the ratio of
midkine protein concentration to thyroglobulin protein
concentration (MK/Tg) in the sample, (e) comparing the ratio of
midkine protein concentration to thyroglobulin protein
concentration determined from the sample with a control, (f)
providing a diagnosis of the growth as a malignant growth after
determining an increased ratio of midkine protein concentration to
thyroglobulin protein concentration determined from the sample as
compared to the control, and (g) performing a thyroidectomy on the
subject when the diagnosis of the growth is that of a malignant
growth, wherein the control is the ratio of midkine protein
concentration to thyroglobulin protein concentration in a benign
growth.
40. The method of claim 39, wherein the growth is a thyroid
nodule.
41. The method of claim 39, wherein the growth is a tumor.
42. The method of claim 39, which further comprises histological or
cytological examination of the growth.
43. The method of claim 39, wherein the immunoassay of (b) is a
sandwich enzyme-linked immunosorbent assay (ELISA) which comprises
a step of contacting the sample with a solution comprising an
antibody that specifically binds to human midline protein.
44. The method of claim 43, wherein the antibody is conjugated to a
label that is biotin, a radionuclide, a fluorescent molecule, a
chemiluminescent molecule, or an enzyme.
45. The method of claim 43, wherein the solution comprises a
polymer.
46. The method of claim 45, wherein the polymer is
poly-L-lysine.
47. The method of claim 46, wherein the concentration of
poly-L-lysine in the solution is 10 .mu.g/mL.
Description
BACKGROUND OF THE INVENTION
[0001] Cancer is a leading cause of death worldwide. Survival rates
for many cancers can be improved by early detection and treatment.
However, for many cancers, diagnostic approaches are highly
invasive, for example, involving surgical biopsy. Less invasive
methods, such as needle biopsies, are often less reliable. More
reliable methods of cancer detection are desired, as are less
invasive methods of detection.
[0002] Midkine, also known as MK, MDK, and NEGF2, is a 13-kDa
heparin-binding growth factor rich in basic amino acids and
cysteine which affects growth, survival, migration, and gene
expression of various target cells. Human midkine is encoded by the
MDK gene. Expression of the MDK gene is induced by retinoic acid or
hypoxia, and suppressed by glucocorticoid. Expression of MDK is
high during midgestation, but low or absent in most adult tissues
(Muramatsu et al., Proc. Jpn. Acad. Ser. B, 86: 410-425
(2010)).
[0003] MDK gene expression is increased in Wilms' tumor specimens
and in human stomach, colon, pancreatic, lung, and esophageal
carcinoma cell lines (Tsutsui et al., Cancer Res., 53: 1281-1285
(1993)). Serum levels of midkine are increased in patients with
esophageal, gastric, duodenal, colon, hepatocellular, bile
duct/gallbladder, pancreatic, thyroid, or lung carcinoma (Ikematsu
et al., Brit. J. Cancer, 83(6): 701-706 (2000)). In situ
hybridization and immunohistochemical analyses demonstrated that
MDK expression and midkine production are increased in human
thyroid papillary carcinoma specimens (Kato et al., Mod. Pathol.,
13(10): 1060-1065 (2000)).
[0004] Improved methods, compositions, and kits for detecting
midkine expression in a biological sample are desired, which may be
useful for diagnostic and prognostic purposes in proliferative
disorders, such as cancer.
BRIEF SUMMARY OF THE INVENTION
[0005] The invention provides a method for diagnosing a growth in a
subject which includes providing a sample of a growth obtained from
a subject, analyzing the sample by an immunoassay to determine the
level of midkine, and comparing the level of midkine determined
from the sample with a control. The level of midkine determined
from the sample relative to the control is diagnostic of a benign
growth or a malignant growth.
[0006] The invention also provides a kit for diagnosing a growth in
a subject. The kit includes an antibody that specifically binds to
midkine, and instructional material for determining, by an
immunoassay, the level of midkine in a sample of a growth obtained
from a subject as compared to a control.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0007] FIG. 1 is a dot plot depicting midkine and thyroglobulin
concentrations in fine-needle aspirate (FNA) samples of
histologically confirmed benign thyroid nodules (open circles) or
papillary thyroid cancer (PTC) (closed circles).
[0008] FIG. 2 is a bar graph depicting midkine to thyroglobulin
ratio (MK/Tg, mean.+-.SEM) in FNA samples of benign thyroid nodules
or malignant papillary thyroid cancer (PTC). Thyroid nodules found
to be benign by cytology only (Benign-C) are shown. separately from
those found to be benign by postoperative histology (Benign-H).
[0009] FIG. 3 is a dot plot depicting the variability of MK/Tg
ratio among individual FNA samples from the same papillary thyroid
cancer. The MK/Tg values of 3-4 individual passages from each of 4
subjects with FTC are shown. Subject 5 had FNA samples both in
vivo, preoperatively (Subject 5-in,) and ex vivo, immediately after
thyroidectomy (Subject 5-ex).
[0010] FIG. 4 is a line graph depicting the absorbance of samples
of recombinant midkine protein of known concentration as detected
by sandwich enzyme-linked immunosorbent assay (EL/SA) performed in
the presence or absence of 10 .mu.g/mL poly-L-lysine (PLL) in the
detection antibody solution.
[0011] FIG. 5 is a dot plot depicting pleiotrophin and
thyroglobulin concentrations in ex viva FNA samples of
histologically confirmed benign thyroid nodules (open circles) or
PTC tissue (closed circles).
[0012] FIG. 6A is a dot plot depicting pleiotrophin to
thyroglobulin (PTN/Tg) ratios in ex viva FNA samples of benign
thyroid nodules (open circles) or PTC tissue (closed circles). FIG.
6B is a bar graph depicting PTN/Tg ratios (mean.+-.SEM) in ex vivo
FNA samples of benign thyroid nodules or FTC tissue, wherein benign
nodules from patients with Graves' disease are separated from total
benign nodules.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The invention provides methods and kits for determining
whether a growth is benign or malignant based upon the level of
midkine present in a sample of the growth.
[0014] In one aspect, the invention provides a method for
diagnosing a growth in a subject. The method comprises (a)
providing a sample of a growth obtained from a subject, (b)
analyzing the sample by an immunoassay to determine the level of
midkine, and (c) comparing the level of midkine determined from the
sample with a control. The level of midkine determined from the
sample as compared with the control is diagnostic of a benign
growth or a malignant growth.
[0015] In another aspect, the invention provides a kit for
diagnosing a growth in a subject. The kit comprises (a) an antibody
that specifically binds to human midkine, and (b) instructional
material for determining the level of midkine by an immunoassay in
a sample of a growth from a subject as compared with a control. The
level of midkine determined from the sample as compared with the
control is diagnostic of a benign growth or a malignant growth.
[0016] The term "growth" as used herein refers to an abnormal mass
of tissue. The "growth" can be a solid mass, a fluid-filled cavity,
or a mixture of solid and fluid components.
[0017] All tumors are growths. The tumor can be a solid tumor or a
hematologic tumor. A solid tumor can be derived from any tissue or
organ, including but not limited to the kidney, liver, cervix,
skin, thyroid, brain, breast, colon, lung, pancreas, prostate,
stomach, ovaries, testicles, urinary bladder, bile duct, or gall
bladder. A solid tumor also can be derived from bone. Hematologic
tumors include leukemia and lymphoma.
[0018] In certain embodiments, the growth is a nodule. The term
"nodule" refers to an abnormal mass present in an otherwise normal
tissue. In some embodiments, the nodule is a thyroid nodule. In
other embodiments, the nodule is a lung nodule.
[0019] In other embodiments, the growth is a "cyst" or an "abscess"
comprised of solid, fluid, or semi-solid components.
[0020] In the context of the invention, the sample can be any
sample taken from a growth present in an organ or tissue of a
subject. The sample can be taken from any suitable region of the
growth, such as a region approximating the center of a growth, or a
region near the periphery of a growth.
[0021] A sample for use in the invention can be taken by any
suitable method capable of collecting cells. The sample can be
obtained from a surgical or open biopsy, or the sample can be
obtained using a needle biopsy. Thus, the sample may be derived
from a "punch," "shave," curettage, fine needle aspiration (FNA),
core needle sample, sentinel lymph node, or excisional biopsy, or
any other method of biopsy known in the art.
[0022] In certain preferred embodiments, the sample is obtained by
FNA. The sample can be all of the material obtained by FNA, or the
sample can be a portion of the material obtained by FNA. For
example, the sample can be a "washout" or "rinse" of the material
remaining in a FNA needle after expression of the contents of the
needle onto a microscope slide for conventional cytological or
histological analyses.
[0023] The subject can be a human or any suitable non-human mammal
such as a mouse, rat, rabbit, cat, dog, pig, sheep, cow, or
primate. In some embodiments, the subject is a non-human
experimental animal model. In some embodiments, the subject is a
primate. Preferably, the subject is a human.
[0024] The immunoassay used to determine the level of midkine in a
sample is not particularly limited. In some embodiments, the
immunoassay is enzyme-linked immunosorbent assay (ELISA), sandwich
ELISA, radioimmunoassay, immunoradiometric assay, gel diffusion
precipitation reaction, immunodiffusion assay, in situ immunoassay
western blot, immunoprecipitation assay, or immunofluorescence
assay. In certain preferred embodiments, the immunoassay is a
"sandwich ELISA" wherein a surface coated with a first antibody
specific for midkine is contacted with a solution containing a
sample under conditions in which a stable complex can form between
the first antibody and the midkine present in the sample, and the
bound midkine is detected with a second antibody specific for
midkine using any suitable means in the art, wherein the detection
of a complex indicates the presence of midkine in the sample.
Methods of performing immunoassays are well-known to those of skill
in the art (see, e.g., Antibodies: A Laboratory Manual, E. Harlow
and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring
Harbor, N.Y., 1988))
[0025] An antibody which "specifically binds" or which is "specific
for" midkine refers to any antibody, or fragment or derivative
thereof, that recognizes at least one antigen or epitope of midkine
and can remain stably bound thereto under standard binding,
washing, and detection conditions utilized in an immunoassay. The
terms "anti-midkine antibody" and "antibody against midkine" as
used herein refer to an antibody which "specifically binds" or
which is "specific for" midkine.
[0026] The antibody specific for midkine can be a polyclonal
antibody, a monoclonal antibody, a chimeric antibody, a single
chain of an antibody, or an Fab fragment that binds to midkine. For
example, the antibody against midkine can be a monoclonal antibody
directed against a single midkine epitope, a combination of
monoclonal antibodies directed against different epitopes of a
single midkine antigenic component, monoclonal antibodies directed
towards epitopes of different midkine antigenic components,
polyclonal antibodies directed towards the same midkine antigen, or
polyclonal antibodies directed towards different midkine
antigens.
[0027] The antibody specific for midkine can be prepared by any
suitable manner.
[0028] A polyclonal antibody can be prepared by immunizing a host
animal, e.g., by injection, with a midkine polypeptide or a
derivative (e.g., fragment or fusion protein) thereof. Suitable
host animals include, but are not limited to, rabbits, mice, rats,
sheep, goats, etc. A midkine polypeptide can be produced
recombinantly or by chemical synthesis, and a fragment or other
derivative or analog thereof, including a fusion protein, can be
used as an immunogen to generate an antibody that recognizes the
midkine polypeptide. In one embodiment, the midkine polypeptide or
fragment thereof can be conjugated to an immunogenic carrier, e.g.,
bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
Adjuvants can be used to increase the immunological response of the
host animal, depending on the host species, including but not
limited to Freund's (complete and incomplete), mineral gels (such
as aluminum hydroxide), surface active substances (such as
lysolecithin), pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and human
adjuvants (such as BCG (bacille Calmette-Guerin) and
Corynebacterium parvum).
[0029] A monoclonal antibody can be prepared by any technique that
provides an antibody by a continuous cell line in culture. These
techniques include, but are not limited, to the hybridoma technique
originally developed by Kohler and Milstein (Nature, 256: 495-497
(1975)), as well as the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., Immunol. Today, 4: 72 (1983);
Cole et al., Proc. Natl. Acad. Sci. USA, 80: 2026-2030 (1983)), and
the EBV hybridoma technique (Cole et al., "The EBV-hybridoma
technique and its application to human lung cancer" in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96
(1985)). The production of monoclonal antibodies by CDR grafting is
described in U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and
5,225,539. Additionally, monoclonal antibodies can be produced in
germ-free animals, as described in international Patent Application
Publication No. WO 19891012690.
[0030] A chimeric antibody can be prepared, for example, by
splicing the genes from a mouse antibody specific for a midkine
polypeptide together with genes from a human antibody of
appropriate biological activity (Morrison et al., J. Bacterial.,
159: 870 (1984); Neuberger et al., Nature, 312: 604-608 (1984); and
Takeda et al., Nature, 314: 452-454 (1985)). Techniques for the
production of single chain antibodies (as described in, for
example, U.S. Pat. Nos. 5,476,786, 5,132,405, and 4,946,778) can be
adapted to produce an antibody against midkine.
[0031] An antibody fragment, which contains the idiotype of the
antibody against midkine, can be generated in any suitable manner,
e.g., using known techniques. Suitable antibody fragments include,
but are not limited to, a F(ab').sub.2 fragment which can be
produced by pepsin digestion of the antibody molecule, a Fab'
fragment which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragment, and a Fab fragment which can be
generated by treating an antibody with papain and a reducing
agent.
[0032] The antibody specific for midkine also can be an
anti-midkine antibody known in the art, such as a commercially
available anti-midkine antibody obtainable from BioVendor (Czech
Republic), Abeam Inc. (Cambridge, Mass.), or R&D Systems Inc.
(Minneapolis, Minn.).
[0033] In the method of the invention, an antibody against midkine,
as bound to midkine present in the sample, is detected so as to
determine the level of midkine. The detection of the antibody
against midkine can be accomplished by any suitable technique known
in the art. In one embodiment, antibody binding is detected by
detecting a label conjugated to the antibody against midkine. In
another embodiment, the antibody binding is detected by binding of
a secondary antibody or reagent to the antibody against midkine,
wherein the secondary antibody is labeled and detected.
[0034] Any suitable label can be utilized so as to determine the
level of midkine present in a sample. Suitable labels include, but
are not limited to, an enzyme, (e.g., alkaline phosphatase,
horseradish peroxidase, etc.), a fluorescent molecule, a
chemiluminescent molecule, a radionuclide, and a dye molecule.
Other reagents and materials can be utilized to facilitation
determination of the level of midkine present in a sample, such as
pretreatment and blocking reagents, amplification reagents (e.g.,
biotin-streptavidin), wash buffers, blocking reagents, and
co-staining reagents. Methods of detecting an antigen-antibody
complexes are well-known to those of skill in the art.
[0035] In some embodiments, the control is a purified midkine
protein produced recombinantly in a host cell. In yet other
embodiments, the control is a purified midkine protein isolated
from cell or tissue which produces midkine endogenously. Thus, a
kit of the invention can include an amount of purified midkine
protein useful as a control, e.g., to construct a standard curve
for comparison with the level of midkine present in a sample. A
control midkine protein can be identical to midkine found in a
human or other mammalian cell or tissue, or the control midkine
protein can be a modified midkine protein, such as a fusion
protein, a truncated protein, or a protein containing amino acid
substitutions.
[0036] In the method of the invention, the level of midkine present
in a sample typically is normalized to the level of a protein or
other substance present in the sample. In some embodiments, the
level of midkine present in a sample is normalized to the level of
a protein encoded by a "housekeeping gene" which is expressed in
the sample. The term "housekeeping gene" is well-known in the art
as referring to a gene expressed at a relatively constant level
during physiological and pathophysiological conditions. A protein
encoded by any housekeeping gene can be used to normalize the level
of midkine present in a sample. Examples of suitable normalization
proteins include, for example, .beta.-actin, Hsp90, and GAPDH. In
some embodiments, the protein used to normalize the level of
midkine is encoded by a gene which is expressed in a
tissue-specific, e.g., organ-specific or tumor-specific, manner.
For example, the level of thyroglobulin can be used to normalize
the level of midkine present in a thyroid nodule or thyroid tumor.
Tissue-specific genes and their protein products are well-known to
those of skill in the art. In other embodiments, the substance used
for normalization represents a set of related molecules, for
example, total protein in the sample. In other embodiments, the
substance is not a protein but another component present in a
sample, such as a nucleic acid, lipid, carbohydrate, or small
organic or non-organic molecule.
[0037] Any suitable method known in the art can be used to
determine the level of a protein used to normalize the level of
midkine in a sample. In some embodiments, the method for
determining the level of a normalization protein in a sample is the
same as the method for determining the level of midkine in the
sample, except that an antibody specific for the normalization
protein is substituted for an anti-midkine antibody.
[0038] The control can be any suitable comparator. In some
embodiments, the control is a sample of normal tissue taken from
the same subject having the growth and desirably the same organ
from which the growth is obtained. For example, if the growth is a
thyroid nodule, the control can be a sample of normal thyroid
tissue taken from the subject with the thyroid nodule. In other
embodiments, the control is a sample of normal tissue taken from
another member of the same species as the subject having the
growth, and desirably the same organ from which the growth is
obtained. For example, if the growth is a thyroid nodule in a human
subject, the control can be a sample of normal thyroid tissue taken
from another human subject. The control also can be a historical
control, such as a level of midkine determined from a prior sample
of a growth obtained from the same subject from which a subsequent
sample of a growth is obtained. The subsequent sample can be
derived from the same growth as a prior sample, or a different
growth. For example, if the growth is a thyroid nodule in a human
subject, the control can be a level of midkine determined from a
prior sample of a thyroid nodule taken from the same human subject.
Preferably, the same method used to normalize the level of midkine
in a sample of a growth is used to normalize the level of midkine
in a control tissue sample.
[0039] In still yet other embodiments, the control is a
predetermined midkine level or predetermined set of midkine levels.
Thus, in the context of the invention, it is not necessary to
perform an assay on a control sample in order to provide a control
midkine level. In some embodiments, the predetermined midkine level
is determined by previous studies of benign growths, malignant
growths, and/or normal tissues in other individuals of the same
species from which a sample of a growth is obtained.
[0040] The control can be a combination of any two or more of the
foregoing controls. Those of skill in the art will understand that
the determination of a suitable control will depend upon several
factors, such as the tissue origin and predicted malignancy of the
growth.
[0041] In an embodiment of the method of the invention, the
determination of an equivalent or decreased level of midkine in a
sample of a growth as compared to a control, especially a benign
control, is diagnostic of a benign growth, whereas the
determination of an increased level of midkine in a sample of a
growth as compared to a known benign control is diagnostic of a
malignant growth. The term "benign" refers to a growth which is
non-cancerous and lacks the ability to metastasize, whereas the
term "malignant" refers to a growth which is cancerous and often
has the ability to metastasize. A malignant growth can be of any
grade of malignancy, e.g., pre-malignant, low grade, intermediate
grade, or high grade. One of ordinary skill in the art understands
that grading systems differ for each type of cancer. All technical
and scientific terms used herein have the meaning commonly
understood by a person skilled in the art to which this invention
belongs, unless explicitly stated otherwise.
[0042] The "threshold" or "cut-off" value for determining whether a
level of midkine determined from a sample is indicative of a benign
growth or a malignant growth is variable and will depend upon
several factors, such as the tissue origin of the growth, number of
samples and controls, and choices of immunoassay and normalization
protein. One of ordinary skill in the art can determine an
appropriate cut-off value empirically based upon any combination of
the foregoing factors.
[0043] In certain embodiments, the method and kit of the invention
are used to determine whether a thyroid growth is benign or
malignant, the normalization protein is thyroglobulin, and the
threshold value is 10 nanograms midkine per milligram
thyroglobulin. Examples of benign thyroid growths include
adenomatoid/colloid nodules, follicular adenomas, and nodules
associated with underlying chronic lymphocytic thyroiditis.
Examples of malignant thyroid growths include papillary thyroid
cancer (PTC), follicular thyroid cancer, medullary thyroid cancer,
and anaplastic thyroid cancer.
[0044] In other embodiments, the method and kit of the invention
are used to diagnose a growth associated with another cancer,
including but not limited to acute lymphoblastic leukemia, acute
myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma,
AIDS-related malignancies, anal cancer, cerebellar astrocytoma,
extrahepatic bile duct cancer, bladder cancer,
osteosarcoma/malignant fibrous histiocytoma, brain stem glioma,
ependymoma, visual pathway and hypothalamic gliomas, breast cancer,
bronchial adenomas/carcinoids, carcinoid tumors, gastrointestinal
carcinoid tumors, carcinoma, adrenocortical, islet cell carcinoma,
primary central nervous system lymphoma, cerebellar astrocytoma,
cervical cancer, chronic lymphocytic leukemia, chronic myelogenous
leukemia, clear cell sarcoma of tendon sheaths, colon cancer,
colorectal cancer, cutaneous t-cell lymphoma, endometrial cancer,
ependymoma, esophageal cancer, Ewing's sarcoma/family of tumors,
extracranial germ cell tumors, extragonadal germ cell tumors,
extrahepatic bile duct cancer, eye cancers, including intraocular
melanoma, and retinoblastoma, gallbladder cancer, gastrointestinal
carcinoid tumor, ovarian germ cell tumor, gestational trophoblastic
tumor, hairy cell leukemia, head and neck cancer, Hodgkin's
disease, hypopharyngeal cancer, hypothalamic and visual pathway
intraocular melanoma, Kaposi's sarcoma, laryngeal cancer, acute
lymphoblastic leukemia, acute myeloid leukemia, chronic
lymphocytic, leukemia, chronic myelogenous leukemia, liver cancer,
non-small cell lung cancer, small cell lung cancer, Hodgkin's
disease, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia,
malignant mesothelioma, malignant thymoma, medulloblastoma,
melanoma, intraocular melanoma, merkel cell carcinoma, metastatic
squamous neck cancer with occult primary, multiple endocrine
neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis
fungoides, myelodysplastic syndrome, chronic myelogenous leukemia,
myeloid leukemia, multiple myeloma, myeloproliferative disorders,
nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, oral cancer, oral cavity and lip cancer,
oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma
of bone, ovarian cancer, ovarian low malignant potential tumor,
pancreatic cancer, paranasal sinus and nasal cavity cancer,
parathyroid cancer, penile cancer, pheochromocytoma, pituitary
tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer,
renal cell (kidney) cancer, transitional cell cancer (e.g. renal
pelvis and ureter), retinoblastoma, rhabdomyosarcoma, salivary
gland cancer, malignant fibrous histiocytoma of bone, soft tissue
sarcoma, sezary syndrome, skin cancer, small intestine cancer,
stomach (gastric) cancer, supratentorial primitive neuroectodermal
and pineal tumors, cutaneous T-cell lymphoma, testicular cancer,
malignant thymoma, gestational trophoblastic tumor, urethral
cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms'
tumor.
[0045] The method and kit of the invention can be used in
conjunction with other methods for diagnosing a growth. Thus, in
some embodiments, a portion of a sample obtained using a needle
biopsy or surgical biopsy can be used in the method of the
invention, while another portion of the same sample can be used in
conventional diagnostic and/or prognostic methods, thereby
decreasing the need for costly and/or invasive procedures in the
subject. In some embodiments, the method and kit of the invention
are used in conjunction with conventional histological or
cytological examination of the growth.
[0046] In some embodiments, the level of midkine present in a
sample level is used to assess the grade of a malignancy, to
predict disease prognosis, to predict the response to a treatment,
and/or to guide the choice of treatment for a growth.
[0047] One advantage of the invention is the high sensitivity of
the midkine assay, which allows for the determination of the
midkine level in a relatively small sample, such as a fine needle
aspirate. In some aspects of the invention, the immunoassay for
detecting midkine levels includes a step of contacting the sample
with a solution comprising (i) an antibody that specifically binds
to human midkine and (ii) a polymer. Without wishing to be bound by
theory, it is believed that the presence of a polymer during
antigen binding increases the affinity or avidity of the
anti-midkine antibody for midkine and/or decreases non-specific
binding of the anti-midkine antibody to other components present in
the sample, thereby increasing the sensitivity of the
immunoassay.
[0048] The amount of anti-midkine antibody present in the solution
is not particularly limited and can be determined empirically by
those of skill in the art. In some embodiments, the antibody
concentration is 0.01 .mu.g/mL or more, e.g., 0.1 .mu.g/mL or more,
1 .mu.g/mL or more, 5 .mu.g/mL or more, 10 .mu.g/mL, or more, or 25
.mu.g/mL or more. Alternatively, or in addition, the antibody
concentration is 100 .mu.g/mL, or less, e.g., 50 .mu.g/mL or less,
25 .mu.g/mL or less, 10 .mu.g/mL or less, 5 .mu.g/mL or less, 2
.mu.g/mL or less, 1 .mu.g/mL or less, or 0.5 .mu.g/mL or less.
Thus, the concentration of anti-midkine antibody present in a
solution during antigen binding can be a concentration bounded by
any two of the above endpoints. For example, the antibody
concentration can be 0.1 .mu.g/mL-5 .mu.g/mL, 1 .mu.g/mL-25
.mu.g/mL, 0.01 .mu.g/mL-1 .mu.g/mL, or 5 .mu.g/mL-10 .mu.g/mL.
[0049] In certain embodiments, the immunoassay involves the use of
two or more distinct anti-midkine antibodies. The concentration of
each anti-midkine antibody for use in a method or kit of the
invention can be selected independently by those of skill in the
art.
[0050] In certain embodiments, the polymer is poly-L-lysine, or a
salt or derivative thereof. The amount of poly-L-lysine present in
the solution is not particularly limited. In some embodiments, the
concentration of poly-L-lysine present in a solution during antigen
binding is 0.1 .mu.g/mL or more, 0.5 .mu.g/mL or more, 1 .mu.g/mL
or more, 5 .mu.g/mL or more, 10 .mu.g/mL or more, or 25 .mu.g/mL or
more. Alternatively, or in addition, the concentration of
poly-L-lysine present in a solution during antigen binding is 1000
.mu.g/mL or less, e.g., 500 .mu.g/mL or less, 250 .mu.g/mL or less,
100 .mu.g/mL or less, 50 .mu.g/mL or less, 20 .mu.g/mL or less, 10
.mu.g/mL or less, or 1 .mu.g/mL or less. Thus, the concentration of
poly-L lysine present in a solution during antigen binding can be a
concentration bounded by any two of the above endpoints. For
example, the concentration of poly-L-lysine present in a solution
during antigen binding can be 0.1 .mu.g/mL-500 .mu.g/mL, 0.5
.mu.g/mL-50 .mu.g/mL, 1 .mu.g/mL-100 .mu.g/mL, or 5 .mu.g/mL-20
.mu.g/mL. In some embodiments, the concentration of poly-L-lysine
present in a solution during antigen binding is 10 .mu.g/mL.
[0051] The molecular weight of the poly-L-lysine is not
particularly limited. In certain embodiments, the poly-L-lysine has
an molecular weight of 0.5 kDa-2 kDa, 1 kDa-5 kDa, 4 kDa-15 kDa, 1
kDa-30 kDa, 30 kDa-70 kDa, 70 kDa-150 kDa, or 150 kDa-300 kDa. The
poly-L-lysine can be in free base form, or the poly-L-lysine can be
in salt form, such as poly-L-lysine hydrochloride or poly-L-lysine
hydrobromide.
[0052] In some embodiments, the solution comprising an antibody
that specifically binds to human midkine contains a polymer which
is a derivative of poly-L-lysine. Suitable derivatives of
poly-L-lysine include, for example, copolymers of poly-L-lysine and
poly-D-lysine, poly-L-lysine conjugated to a protein, and
copolymers of poly-L-lysine and poly(ethylene glycol).
[0053] In other embodiments, the solution comprising an antibody
that specifically binds to human midkine contains a polymer having
physiochemical properties similar to poly-L-lysine. Thus, in
certain embodiments of the invention, the solution comprising an
antibody that specifically binds to human midkine contains another
positively charged, water soluble polymer, such as poly-D-lysine or
polyarginine.
[0054] Although the precise size of a sample is not limiting, a
sample for use in the method and kit of the invention can contain
as little as 0.1 mg total protein, e.g., 0.5 mg total protein or
more, 0.0 mg total protein or more, 0.8 mg total protein or more,
1.0 mg total protein or more, 1.5 mg total protein or more, 3.0 mg
total protein or more, 10 mg protein or more, or 25 mg protein or
more. Alternatively, or in addition, the sample can contain 200 mg
protein or less, e.g., 100 mg total protein or less, 50 mg total
protein or less, 30 mg total protein or less, 20 mg total protein
or less, 7.5 mg total protein or less, 1 mg total protein or less,
or 0.75 mg total protein or less. Thus, the amount of total protein
present in a sample for use in the method and kit of the invention
can be an amount bounded by any two of the above endpoints. For
example, the amount of total protein present in a sample for use in
the method and kit of the invention can be 0.1 mg-20 mg, 0.5 mg-7.5
mg, 1.0 mg-100 mg, 3.0 mg-30 mg, or 10 mg-200 mg.
[0055] A kit according to the invention comprises at least two
components, namely, an anti-midkine antibody and instructional
material. In certain embodiments, the kit includes two or more
distinct anti-midkine antibodies. The instructional material
includes instructions for (al determining the level of midkine, in
a sample, and (b) interpreting the results of the determination.
Preferably, the instructional material also includes instructions
for determining the level of midkine in a control and/or provides
one or more control levels (e.g., threshold values) for
midkine.
[0056] In some embodiments, the instructional material recites a
step of contacting the sample with a solution comprising an
anti-midline antibody and a polymer. In certain embodiments, the
polymer is poly-L-lysine, or a salt or derivative thereof, as
described above in the context of the methods of the invention.
[0057] In certain embodiments, the instructional material includes
instructions for determining the level of midkine in a sample
obtained from a thyroid growth, and diagnosing whether the thyroid
growth is benign or malignant.
[0058] The kit of the invention can be used in diagnosing a growth
obtained from any subject, particularly a mammal. Preferably the
mammal is a human. In other embodiments, the mammal can be a mouse,
rat, rabbit, cat, dog, pig, sheep, cow, primate, or another
mammal.
[0059] The sample and the control can be any sample or control
suitable for use in the method of the invention as described
above.
[0060] Pleiotrophin, encoded by the PTN gene in humans, is closely
related to midkine by amino acid sequence homology and structural
similarity. Pleiotrophin is also known as heparin-binding brain
mitogen (HBBM), heparin-binding growth factor 8 (HBGF-8), neurite
growth-promoting factor 1 (NEGF1), heparin affinity regulatory
peptide (HARP), and heparin binding growth associated molecule
(HB-GAM). Together, midkine and pleiotrophin form a family of
heparin-binding proteins often referred to as the neurite
growth-promoting factor (NEGF) family (Muramatsu et al., Proc. Jpn.
Acad., Ser. B, 86: 410-425 (2010)).
[0061] PTN gene expression is increased in several cancers,
including pancreatic carcinoma, choriocarcinoma, neuroblastoma, and
melanoma (Muramatsu, J. Biochem., 132, 359-371 (2002)). In
addition, serum levels of pleiotrophin are increased in patients
with pancreatic and colon carcinoma (Muramatsu, ibid).
[0062] Thus, the invention also provides methods and kits for
determining whether a growth is benign or malignant based upon the
level of pleiotrophin present in a sample of the growth. In one
aspect, the invention provides a method for diagnosing a growth in
a subject. The method comprises (a) providing a sample of a growth
obtained from a subject, (b) analyzing the sample by an immunoassay
to determine the level of pleiotrophin, and (c) comparing the level
of pleiotrophin determined from the sample with a control. The
level of pleiotrophin determined from the sample as compared with
the control is diagnostic of a benign growth or a malignant
growth.
[0063] In another aspect, the invention provides a kit for
diagnosing a growth in a subject. The kit comprises (a) an antibody
that specifically binds to human pleiotrophin, and (b)
instructional material for determining the level of pleiotrophin by
an immunoassay in a sample of a growth from a subject as compared
with a control. The level of pleiotrophin determined from the
sample as compared with the control is diagnostic of a benign
growth or a malignant growth.
[0064] The embodiments of the method and kit for determining
whether a growth is benign or malignant based upon the level of
pleiotrophin present in a sample of the growth are similar to the
embodiments of the method and kit described hereinabove with
respect to midkine, except that an antibody specific for midkine is
substituted for an antibody specific for pleiotrophin.
[0065] An antibody specific for pleiotrophin can be generated by
any suitable manner known in the art, such as those described
herein with respect to the generation of anti-midkine antibodies.
The antibody specific for pleiotrophin also can be an
anti-pleiotrophin antibody known in the art, such as a commercially
available anti-pleiotrophin antibody obtainable from EMD Millipore
(Billerica, Mass.) or R&D Systems Inc. (Minneapolis, Minn.). In
certain embodiments, the anti-pleiotrophin antibody is a mouse
monoclonal anti-pleiotrophin antibody known as 3B10, which is
commercially available.
[0066] In some embodiments, the method or kit for determining
whether a growth is benign or malignant based upon the level of
pleiotrophin present in a sample of the growth includes the use of
a polymer, such as poly-L-lysine, as described hereinabove with
respect to the method and kit based upon midkine. In other
embodiments, the method or kit for determining whether a growth is
benign or malignant based upon the level of pleiotrophin present in
a sample of the growth does not include the use of a polymer.
[0067] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
[0068] This example demonstrates a method for diagnosing a thyroid
nodule by determining the level of midkine in a sample of a thyroid
nodule by an immunoassay.
[0069] Samples of thyroid tissue were obtained from adult subjects
with thyroid nodules who underwent percutaneous fine-needle
aspiration (FNA) (n=41), thyroidectomy (n=23), or percutaneous FNA
and thyroidectomy (n=9). To obtain the samples, a 25-gauge needle
was repeatedly passed into a nodule (either in vivo by percutaneous
FNA, or ex vivo immediately after thyroidectomy to mimic
percutaneous FNA), and the needle was rinsed with 500 .mu.L
phosphate buffered saline (PBS) containing 1% bovine serum albumin
(BSA) either before or after expressing a portion of the sample
onto a microscope slide for conventional cytological analysis. The
rinse (also referred to as a "washout") was aliquotted and stored
at -80.degree. C. until immunoassay.
[0070] A midkine (MK) sandwich ELISA immunoassay was performed
using a commercial kit (BioVendor, Czech Republic) with
modifications. 50 .mu.L of thyroid washout was diluted in 200 .mu.L
of TBSTA (Tris-buffered saline, 50 mM Tris-HCL, pH 8, 0.15 M NaCl,
0.5% Tween 20, 1% BSA). 100 .mu.L of the diluted samples was
transferred in duplicate into a 96-well plate, which was pre-coated
by the manufacturer with an anti-midkine capture antibody. The
plate was incubated at 37.degree. C. for 2 hours without shaking
and then washed 3 times with 0.35 mL per well of the washing buffer
provided in the kit. After tapping the inverted plate to remove
residual fluid, 10 .mu.g/mL of poly-L-lysine (Sigma-Aldrich, St.
Louis, Mo.) in water was added to the biotin-labeled antibody
solution provided with the kit, and then 100 .mu.L of this solution
was added to each well. The plate was incubated at room temperature
for 1 hour, with shaking at 300 RPM on an orbital microplate
shaker. Then the wells were washed 5 times with 0.35 mL per well of
the washing buffer provided in the kit. After tapping, 100 .mu.L of
streptavidin-HRP conjugate solution provided with the kit was added
to each well. The plate was incubated at room temperature for 30
minutes, with shaking at 300 RPM on an orbital microplate shaker.
After washing 5 times with washing buffer and tapping, the
substrate solution provided with the kit was added to each well.
The plate was covered with aluminum foil and incubated for 7
minutes at room temperature. Color development was stopped by
adding 100 .mu.L per well of the stop solution provided with the
kit. The absorbance of each well at 450 nm was measured using a
microplate reader. Intra-assay CV was 3.4% for high concentration
and 5.2% for low concentration. The limit of detection was 8.7
pg/mL midkine. The absolute concentration of midkine in a sample
was calculated by comparison to a control, which consisted of
multiple dilutions of recombinant human midkine of known
concentrations.
[0071] A thyroglobulin (Tg) assay also was performed on an aliquot
of each washout. Briefly, an aliquot of each washout was diluted
10-fold in normal saline, and the thyroglobulin concentration was
measured with a chemiluminescent immunometric assay (Immulite
2000XPi, Siemens, UK) according to the manufacturer's instructions.
Thyroglobulin reagent (Siemens Healthcare Diagnostics, USA) was
used to further dilute each sample, if needed.
[0072] The midkine concentrations or the midkine to thyroglobulin
ratios (MK/Tg) of multiple needle passes were averaged for each
subject. Statistical analysis was performed using SigmaPlot
software, version 12. The relationship between midkine and
thyrogloblulin concentrations was analyzed by regression. Midkine
and MK/Tg were compared in benign versus malignant nodules by
T-test. For this analysis, all FNA samples (separate passes of the
needle) were averaged to obtain a single mean for each subject. For
subjects with both in viva and ex viva samples, only the in viva
values were included, which are more relevant clinically. P
values<0.05 were considered significant.
[0073] For normal thyroid tissue samples (obtained ex viva from
thyroid gland distant from any nodule), washout of the FNA needle
contained very low concentrations of MK (0.03.+-.0.01 ng/mL). MK
concentrations were also low in samples obtained ex viva from
nodules that were benign by surgical pathology (0.07.+-.0.02 ng/mL)
and samples obtained in viva from nodules with benign cytology
(0.02.+-.0.01 ng/mL). In the subjects with surgically confirmed
benign nodules, MK concentrations were similarly low in
adenomatoid/colloid nodules (0.027.+-.0.016 ng/mL, n=12),
follicular adenomas (0.079.+-.0.073 ng/mL, n=3), and benign nodules
with underlying chronic lymphocytic thyroiditis (0.008.+-.0.007
ng/mL, n=5). MK concentrations from subjects with Graves' disease
also were low (0.062.+-.0.013 ng/mL, n=3). In contrast, MK
concentrations in samples from papillary thyroid cancer (PTC)
showed higher levels than in benign nodules (0.72.+-.0.27 ng/mL
versus 0.03.+-.0.01 p=0.006), irrespective of whether the samples
were obtained in viva or ex viva (FIG. 1). Conversely,
thyroglobulin concentrations were lower in samples of FTC than in
samples of benign nodules (7540.+-.4600 ng/mL versus
110000.+-.28900 ng/mL, p<0.001) (FIG. 1).
[0074] The results of this example demonstrate that midkine protein
levels in FNA washout samples are higher in papillary thyroid
cancer than in normal thyroid tissue or benign thyroid nodules.
EXAMPLE 2
[0075] This example demonstrates a method for diagnosing a thyroid
nodule by determining the ratio of midkine to thyroglobulin in a
sample of a thyroid nodule.
[0076] Midkine to thyroglobulin (MK/Tg) ratios were calculated
based upon the results of the assays described in Example 1. MK
concentration was positively associated with thyroglobulin
concentrations in FNA washout samples from both benign and
malignant nodules (R.sup.2=0.09, p<0.001 and R.sup.2=0.53,
p<0,001, respectively). This correlation likely occurs because
both MK and thyroglobulin concentrations in the washout fluid are
dependent on the amount of thyroid tissue present in the sample.
Therefore, to correct for the amount of thyroid tissue, midkine
levels were normalized to thyroglobulin levels by calculating the
MK/Tg ratio. In subjects with both in vivo and ex vivo samples, the
MK/Tg ratio was similar in the two types of samples (1.81.+-.0.95
ng/mg versus 1.74.+-.0.70 ng/mg, respectively for benign nodules,
p=NS).
[0077] Samples from normal thyroid tissue and Graves' disease
showed low midkine to thyroglobulin ratios. Similarly low values
were seen for benign nodules, which included adenomatoid/colioid
nodules, follicular adenomas, and benign nodules within a thyroid
affected by chronic lymphocytic thyroiditis. In contrast, the MK/Tg
ratio was far higher in papillary thyroid cancer (PTC) samples than
in benign nodules (403.+-.285 ng/mL versus 1.51.+-.0.55 ng/mg,
P<0.001, the subjects with a histological diagnosis) (FIG. 2).
The MK/Tg ratio tended to increase with nodule size in PTC, but the
relationship did not reach statistical significance (R.sup.2=0.51,
P=0.09).
[0078] For the preceding analysis, all FNA samples (separate passes
of the needle) were averaged tor each subject. To assess the
sampling variability in the MK/Tg ratio, individual FNA samples
from the same malignant lesion were compared. There was a
reasonably close agreement among individual samples, and all
individual MK/Tg ratios were substantially higher than the MK/Tg
ratios determined from benign lesions (FIG. 3).
[0079] The results of this example demonstrate that midkine to
thyroglobulin ratios in FNA washout samples are higher in papillary
thyroid cancer than in normal thyroid tissue or benign thyroid
nodules.
EXAMPLE 3
[0080] This example demonstrates a method of increasing the
sensitivity of an immunoassay for determining the level of midkine
in a sample.
[0081] A midkine sandwich ELISA immunoassay was performed using a
commercial kit (BioVendor, Czech Republic) as described in Example
1, except that recombinant human midkine at a concentration of 0,
0.033, 0.1, 0.3, 0.9 ng/mL, was prepared in 200 .mu.L of TBSTA
instead of a thyroid washout. Increasing concentrations of
recombinant human midkine produced increasing signal in the
sandwich ELISA (FIG. 4). Addition of 10 .mu.g/mL poly-L-lysine
(PLL) to the detection antibody solution markedly increased the
overall absorbance values at each midkine concentration as well as
the slope of the response curve (FIG. 4).
[0082] The results of this example demonstrate that the addition of
poly-L-lysine to a solution containing an antibody specific for
midkine increases the sensitivity of an immunoassay.
EXAMPLE 4
[0083] This example demonstrates a method for diagnosing a thyroid
nodule by determining the level of pleiotrophin in a sample of a
thyroid nodule by an immunoassay.
[0084] Samples of thyroid tissue were obtained from adult subjects
with thyroid nodules who underwent thyroidectomy (n=31). After the
thyroid was excised, the nodules of interest were identified by the
surgeon and pathologist, and the selected nodules with surrounding
tissues were bisected for procurement. 25 benign nodules from 23
subjects and 10 nodules with PTC from 8 subjects were identified by
histological analyses. No follicular, medullary, or anaplastic
thyroid cancers were identified by histological analyses. 2
subjects had underlying Graves' disease. 1 subject with benign
nodules had underlying chronic lymphocytic thyroiditis.
[0085] Ex vivo fine-needle aspiration (FNA) was performed by
repeatedly passing a 25-gauge needle into a nodule to mimic
percutaneous FNA. The needle was rinsed with 500 .mu.L phosphate
buffered saline (PBS) containing 1% bovine serum albumin (BSA) to
obtain washout samples, which were aliquotted and stored at
-80.degree. C. until immunoassay. Multiple ex vivo FNA samples
(mean of 3.0 passes) were obtained per nodule. Normal thyroid
tissue samples were obtained from 11 subjects by performing ex vivo
FNA on normal thyroid tissue adjacent to benign or malignant
nodules.
[0086] A pleiotrophin sandwich ELISA immunoassay was performed
using a mouse anti-pleiotrophin monoclonal antibody (3B10,
available from EMD Millipore, Billerica, Mass.) and a biotinylated
anti-human pleiotrophin goat IgG (available from R&D Systems,
Inc., Minneapolis, Minn.). The 3B10 antibody was diluted to 0.5
.mu.g/mL in PBS, and 100 .mu.L/well was incubated in a 90-well
plate at 4.degree. C. overnight. The wells were washed 3 times with
250 .mu.L per well PBST (PBS, 0.05% TWEEN.TM. 20). The wells were
blocked with 250 .mu.L per well of PBS containing 3% BSA and 0.2%
TWEEN.TM. 20 for 2 hours at 4.degree. C. Without washing, the plate
was inverted and dried by tapping vigorously against a paper towel.
100 .mu.L of thyroid washout sample was diluted in 200 .mu.L PBSTA
(PBS, 1% BSA, 0.5% TWEEN.TM. 20). 100 .mu.L of the diluted sample
was transferred, in duplicate, into the plate. The plate was
incubated with gentle agitation at room temperature for 2 hours,
and then washed 3 times with 250 per well of PBST. After tapping
the inverted plate to remove residual fluid, the biotinylated
anti-human pleiotrophin goat IgG was added at a concentration of
500 ng/mL and diluted in a 0.9% normal saline containing 5.7 mEq/L
calcium chloride and 0.5% BSA, at pH 6. The plate was incubated
with gentle agitation at room temperature for an hour. The wells
were washed 5 times with 250 .mu.L of PBST per well. After tapping
the inverted plate to remove residual fluid, 100 .mu.L
streptavidin-HRP conjugate solution (Thermo Scientific, Rockford,
Ill.) was added at a concentration of 25 ng/mL in PBS to each well,
and the plate was incubated at room temperature for 30 minutes with
gentle agitation. After washing 5 times with PBST, 100 .mu.L of
3,3',5,5'-tetramethylbenzidine (TMB) was added to each well. The
plate was covered with aluminum foil and incubated for 7 minutes at
room temperature. Color development was stopped by adding 100 .mu.L
of stop solution. The absorbance of each well at 450 nm was
measured using a microplate reader. The intra-assay CV was 5.1% for
high concentration and 7.3% for low concentration. The inter-assay
CV was 6.4%. The absolute concentration of pleiotrophin in a sample
was calculated by comparison to a control, which consisted of
multiple dilutions of recombinant human pleiotrophin of known
concentrations.
[0087] A thyroglobulin (Tg) assay also was performed on an aliquot
of each washout. Briefly, 50 .mu.L of thyroid washout sample was
diluted 10-fold in normal saline, and the thyroglobulin
concentration was measured with a chemiluminescent immunometric
assay (Immulite 2000XPi, Siemens, UK) according to the
manufacturer's instructions. Thyroglobulin reagent (Siemens
Healthcare Diagnostics, USA) was used to further dilute each
sample, if needed.
[0088] The pleiotrophin (PTN) concentrations or the pleiotrophin to
thyroglobulin ratios (PTN/Tg) of multiple needle passes were
averaged to obtain a single mean value for each nodule. Statistical
analysis was performed using SigmaPlot software, version 12. The
relationship between pleiotrophin and thyrogloblulin concentrations
was analyzed by Pearson correlation after log transformation of
both variables. Pleiotrophin and PTN/Tg were compared in benign
versus malignant nodules by T-test. P values<1.05 were
considered significant.
[0089] The mean concentration of PTN in normal thyroid tissue was
29.+-.13 pg/mL. PTN concentrations in samples from PTC tissue were
higher than in samples from benign nodules (85.+-.25 pg/mL versus
35.+-.11 pg/mL, mean.+-.SEM, p=0.01) (Table and FIG. 5).
Conversely, thyroglobulin concentrations in samples from PTC tissue
were lower than in samples from benign nodules (59.4.+-.24 .mu.g/mL
versus 664.+-.146 .mu.g/mL, p=0.008) (Table and FIG. 5).
[0090] The PTN and PTN/Tg values, along with the pathology results,
are set forth in the following Table.
TABLE-US-00001 TABLE Pleiotrophin and pleiotrophin/thyroglobulin
ratio with pathology results PTN PTN/Tg ratio Surgical Pathology -
(pg/mL) (pg/mg) Benign N = 25 Mean .+-. SD Mean .+-. SD
Adenomatoid/Nodular 19 38 .+-. 65 118 .+-. 261 hyperplasia
Autoimmune thyroiditis 1 0 0 Follicular adenoma 3 15 .+-. 21 54
.+-. 76 Focal epithelial 1 74 2334 hyperplasia/Graves Nodular
hyperplasia/Graves 1 40 1639 All benign nodules 25 35 .+-. 11 357
.+-. 145 Surgical Pathology - PTC N = 8 Classic 2 86 .+-. 48 19500
.+-. 26700 Tall cell 3 115 .+-. 109 10900 .+-. 8250 Follicular
variant 2 61 .+-. 23 724 .+-. 186 Unclassified 1 20.6 2949 All
malignant nodules 8 85 .+-. 25 9490 .+-. 4730
[0091] The results of this example demonstrate that pleiotrophin
levels in FNA washout samples are higher in papillary thyroid
cancer tissue than in normal thyroid tissue or benign thyroid
nodules.
EXAMPLE 5
[0092] This example demonstrates a method for diagnosing a thyroid
nodule by determining the ratio of pleiotrophin to thyroglobulin in
a sample of a thyroid nodule.
[0093] Pleiotrophin to thyroglobulin (PTN/Tg) ratios were
calculated based upon the results of the assays described in
Example 4. The mean PTN/Tg ratio in normal thyroid tissue was
2950.+-.888 pg/mg. The PTN/Tg ratio values were low in benign
nodules, including adenomatoid colloid nodules, follicular
adenomas, and benign nodules within a thyroid affected by chronic
lymphocytic thyroiditis (FIGS. 6A and 6B). The PTN/Tg ratio values
were higher in papillary thyroid cancer tissue, than in benign
nodules (9490.+-.4730 pg/mg versus 357.+-.145 pg/mg, p<0.001)
(FIGS. 6A and 6B). The PTN/Tg ratio did not appear to correlate
with nodule size. Samples from benign nodules obtained from
patients with Graves' disease showed high pleiotrophin to
thyroglobulin ratios (FIG. 6B).
[0094] The results of this example demonstrate that pleiotrophin to
thyroglobulin ratios in FNA washout samples are higher in papillary
thyroid cancer tissue than in benign thyroid nodules.
[0095] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0096] The use of the terms "a" and "an" and "the" and "at least
one" and similar referents 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. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate 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 unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0097] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as 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.
* * * * *