U.S. patent application number 09/728771 was filed with the patent office on 2001-03-29 for diagnostic modified-nucleoside detection assay.
Invention is credited to Grutsch, James F., Moskal, Joseph R..
Application Number | 20010000078 09/728771 |
Document ID | / |
Family ID | 21987392 |
Filed Date | 2001-03-29 |
United States Patent
Application |
20010000078 |
Kind Code |
A1 |
Moskal, Joseph R. ; et
al. |
March 29, 2001 |
Diagnostic modified-nucleoside detection assay
Abstract
The present invention relates to an assay for the detection of
modified nucleoside levels in a patient. Detection of the modified
nucleoside levels in a patient having a disease such as cancer
allows for the progression of the disease to be followed and
therapeutic regimens to be altered. Such an assay is particularly
useful in following the response of cancer patients to
chemotherapeutic treatment.
Inventors: |
Moskal, Joseph R.; (Chicago,
IL) ; Grutsch, James F.; (Wilmette, IL) |
Correspondence
Address: |
McDonnell Boehnen Hulbert & Berghoff
300 S. Wacker Drive
Chicago
IL
60606
US
|
Family ID: |
21987392 |
Appl. No.: |
09/728771 |
Filed: |
December 1, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09728771 |
Dec 1, 2000 |
|
|
|
09053913 |
Apr 2, 1998 |
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Current U.S.
Class: |
436/501 ;
435/6.16; 435/7.1 |
Current CPC
Class: |
G01N 33/57488 20130101;
G01N 2800/52 20130101; G01N 33/5308 20130101; Y10T 436/143333
20150115 |
Class at
Publication: |
436/501 ; 435/6;
435/7.1 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Claims
We claim:
1. A method for determining the effectiveness of a treatment
regimen for treating a condition such as cancer comprising
determining the concentration of a modified nucleoside in a bodily
fluid of a patient suspected of having cancer wherein an increased
or decreased level of modified nucleoside relative to the level of
modified nucleoside detected in the bodily fluid isolated prior to
treatment indicates the effectiveness of the treatment regimen.
2. A method of claim 1 wherein said modified nucleoside is selected
from the group consisting of psueouridine (.psi.), 7-methylinosine
(m.sup.7I), and 1-methyladenosine (m.sup.1A).
3. A method of claim 1 wherein psueouridine (.psi.),
7-methylinosine (m.sup.7I), and 1-methyladenosine (m.sup.1A) are
detected.
4. A method of claim 1 wherein said condition is brain cancer.
5. A method of claim 1 wherein said condition is glioma or
meningioma.
6. A method for determining the effectiveness of a treatment
regimen for treating a condition such as cancer comprising: a)
adding to a bodily fluid of a patient to be treated a first
antibody having immunological reactivity to a modified nucleoside
comprising a label; b) contacting the bodily fluid of step (a) with
a solid surface to which a second antibody specific to a modified
nucleoside is bound under conditions suitable to the formation of a
complex of said modified nucleoside with said labeled antibody and
the antibody bound to the solid surface; and c) detecting the
labeled antibody; wherein detection of the labeled antibody
indicates the presence of a modified nucleoside within the
biological fluid.
7. A method of claim 1 wherein said modified nucleoside is selected
from the group consisting of psueouridine (.psi.), 7-methylinosine
(m.sup.7I), and 1-methyladenosine (m.sup.1A).
8. A method of claim 1 wherein psueouridine (.psi.),
7-methylinosine (m.sup.7I), and 1-methyladenosine (m.sup.1A) are
detected.
9. A method of claim 1 wherein said condition is brain cancer.
10. A method of claim 1 wherein said condition is glioma or
meningioma.
11. A method of claim 5 wherein said solid surface support is a
dipstick.
12. A method for diagnosing cancer comprising determining the
concentration of a modified nucleoside in a bodily fluid of a
patient suspected of having cancer wherein an increased or
decreased level of modified nucleoside relative to the level of
modified nucleoside detected in the bodily fluid of a patient that
does not have cancer indicates the presence of cancer.
13. A method of claim 12 wherein said modified nucleoside is
selected from the group consisting of psueouridine (.psi.),
7-methylinosine (m.sup.7I), and 1-methyladenosine (m.sup.1A).
14. A method of claim 12 wherein psueouridine (.psi.),
7-methylinosine (m.sup.7I), and 1-methyladenosine (m.sup.1A) are
detected.
15. A method of claim 12 wherein said condition is brain
cancer.
16. A method of claim 12 wherein said condition is glioma or
meningioma.
17. A method for diagnosing cancer comprising: a) adding to a
bodily fluid of a patient to be treated a first antibody having
immunological reactivity to a modified nucleoside comprising a
label; b) contacting the bodily fluid of step (a) with a solid
surface to which a second antibody specific to a modified
nucleoside is bound under conditions suitable to the formation of a
complex of said modified nucleoside with said labeled antibody and
the antibody bound to the solid surface; and c) detecting the
labeled antibody; wherein detection of the labeled antibody
indicates the presence of cancer.
18. A method of claim 17 wherein said modified nucleoside is
selected from the group consisting of psueouridine (.psi.),
7-methylinosine (m.sup.7I), and 1-methyladenosine (m.sup.1A).
19. A method of claim 17 wherein said condition is a brain
cancer.
20. A method of claim 17 wherein said solid surface support is a
dipstick.
Description
FIELD OF THE INVENTION
1. The present invention relates to an assay for the detection of
modified nucleoside levels in a patient. Detection of the modified
nucleoside levels in a patient having a disease such as cancer
allows for the progression of the disease to be followed and
therapeutic regimens to be altered. Such an assay is particularly
useful in following the response of cancer patients to
chemotherapeutic treatment.
BACKGROUND OF THE INVENTION
2. Ribonucleic acids (RNA) have central roles in all aspects of
cellular metabolism. Protein synthesis is dependent upon the
coordinated interactions of transfer RNA (tRNA), ribosomal RNA
rRNA), and messenger RNA (mRNA). The cellular roles of RNA are
complex, as they function as structural elements (rRNA), catalytic
components (in RNA processing), informational molecules (mRNA), and
as a direct link between information contained in DNA and in
proteins (tRNA). Gene activation is dependent not only on specific
mRNA transcription but also on its correct splicing, capping and
processing to create a functional mRNA. Consequently, in disease
states such as cancer, where abnormal cell growth occurs, it is not
unexpected that perturbations are indicative of metabolic
alterations that can be exploited for cancer diagnosis or
therapy.
3. RNA contains a variety of modified nucleosides, which are
enzymatically modified post-transcriptionally by a variety of
modification enzymes. Over 50 chemically distinct modifications
have been described, each requiring a different enzyme. In humans
and other animals normal RNA turnover and breakdown creates free
modified nucleosides. These modified nucleosides collect in the
circulation from which they are filtered and concentrated by the
kidneys into the urine for excretion. Consequently, all urine
contains levels of modified nucleosides that reflect RNA
degradation in the organism.
4. Numerous reports have correlated increased rates of modified
nucleoside urinary excretion with cancer and other disease states.
Examples of such cancers include leukemias (Trweyn and Grever.
1986. CRC Crit. Rev. Clin. Lab. Sci., 24:71-93), chronic
myelogenous leukemia (Heldman, et al. 1983. J. Lab. Clin. Med.,
101:783-792), adult acute leukemia (Heldman, et al. 1983. Blood,
61:291-296), childhood acute lymphoblastic leukemia (Heldman, et
al. 1983. J. Nat. Can. Inst., 71: 269-273), bronchogenic carcinoma
(Rasmuson, et al. 1983. Acta Radiolog. Oncology. 22: 209-214),
mammary and colorectal carcinoma (Rasmusen and Bjork, 1985. Bull.
Mol. Biol. Med., 10:143-154), hepatocellular carcinoma (Tamura.
1986. Cancer, 57:1571-1575), small cell lung cancer (Tamura, 1986.
Clin. Chem. Acta, 154:125-132) ovarian cancer (Oerlemans and Lange,
1986.), malignant lymphomas (Rasmuson and Bjork, 1983. ),
nasopharyngeal carcinoma (Trewyn, 1982. Cancer 49: 2513-2517), and
malignant mesothehoma (Fishbein, et al, 1983. Cancer Res.
43:2971-2974).
5. The monitoring of urinary modified nucleoside levels has not
been demonstrated to be useful for cancer diagnosis, to stage
disease, to determine the efficacy of a treatment protocol or to
monitor recurrence of cancers in remission. The present application
provides one skilled in the art with the reagents and methodologies
required to efficiently measure levels of modified nucleosides in a
patient as a diagnostic or predictive assay useful in designing
treatment protocols.
BRIEF DESCRIPTION OF THE FIGURES
6. FIG. 1. Urinary 1-methyladenosine levels in patients with
various cancers.
7. FIG. 2. GBM survival relates to 1-methyladenosine levels.
8. FIG. 3. Midgrade glioma survival and 1-methyladenosine
levels.
SUMMARY OF THE INVENTION
9. The present invention relates to a method for detecting modified
nucleosides in a biological sample such as a bodily fluid. The
methods provided herein are useful for detecting a modified
nucleoside such as psueouridine (.psi.), 7-methylinosine
(m.sup.7I), and 1-methyladenosine (m.sup.1A). It is an object of
the invention to provide a method for determining the effectiveness
of a treatment protocol for treating a disease such as cancer. It
is a further object of the invention to provide an in vitro and an
in vivo assay for determining the effectiveness of a treatment
protocol for treating a disease such as cancer. It is also an
object of the present invention to provide a method of diagnosing
or detecting cancer.
10. In one embodiment, a method for determining the effectiveness
of a treatment protocol comprising determining the concentration of
a modified nucleoside in the biological fluid of a patient is
provided.
11. In another embodiment, an assay is provided wherein the
effectiveness of a particular treatment protocol may be determined
in vitro using a cell line or primary tumor cells or in vivo using
an animal model.
12. In another embodiment, a method for determining the
effectiveness of a treatment protocol comprising contacting a
bodily fluid from a patient with a labeled antibody that is
specific to a modified nucleoside and detecting the labeled
antibody, thus indicating the presence of a modified nucleoside in
the bodily fluid.
DETAILED DESCRIPTION OF THE INVENTION
13. Within this application, unless otherwise stated, the
techniques utilized may be found in any of several well-known
references such as: Molecular Cloning: A Laboratory Manual
(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene
Expression Technology (Methods in Enzymology, Vol. 185, edited by
D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to
Protein Purification" in Methods in Enzymology (M. P. Deutshcer,
ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to
Methods and Applications (Innis, et al. 1990. Academic Press, San
Diego, Calif.), Culture of Animal Cells: A Manual of Basic
Technique, 2.sup.nd Ed. (R. I. Freshney, 1987. Liss, Inc. New York,
N.Y.), and Gene Transfer and Expression Protocols, pp. 109-128, ed.
E. J. Murray, The Humana Press Inc., Clifton, N.J.).
14. The reagents and methodologies of the present invention may be
utilized to prevent, detect, or treat a variety of disorders in
which increased levels of modified nucleosides are produced by the
affected cells. An example of such a disorder is cancer. Cancer is
defined herein as any cellular malignancy for which a loss of
normal cellular controls results in unregulated growth, lack of
differentiation, and increased ability to invade local tissues and
metastasize. Cancer may develop in any tissue of any organ at any
age. Cancer may be an inherited disorder or caused by environmental
factors or infectious agents; it may also result from a combination
of these. For the purposes of utilizing the present invention, the
term cancer includes both neoplasms and premalignant cells. A
specific example of utilization of the present invention to treat
brain cancer is provided herein. Brain cancer is defined herein as
any cancer involving a cell of neural origin. Examples of brain
cancers include but are not limited to intracranial neoplasms such
as those of the skull (i.e., osteoma, hemangioma, granuloma,
xanthoma, osteitis deformans), the meninges (i.e., meningioma,
sarcoma, gliomatosis), the cranial nerves (i.e., glioma of the
optic nerve, schwannoma), the neuroglia (i.e., gliomas) and
ependyma (i.e., ependymomas), the pituitary or pineal body (i.e.,
pituitary adenoma, pinealoma), and those of congenital origin
(i.e., craniopharygioma, chordoma, germinoma, teratoma, dermoid
cyst, angioma, hemangioblastoma) as well as those of metastatic
origin.
15. It has been shown that detection of modified nucleosides in
urine is useful for diagnosis and prognostic evaluation of certain
cancers such as leukemia (U.S. Pat. No. 4,687,733). It has been
found that myelogenous leukemia, acute lymphoblastic leukemia and
chronic myelogenous leukemia may be distinguished by determination
of 1-methylinosine levels in a patient's urine (Heldman et al.
1983. Blood 61: 291-296; Heldman, et al. 1983. J. Lab. Clin. Med.
101: 783-792). The '733 patent demonstrated that detection of
1-methylinosine was positively correlated with the percentage of
blast cells in a patient's bone marrow aspirate. The levels of
1-methylguanosine, pseudouridine, 1-methyladenosine, or
5-methylcytidine did not correlate with blast cell numbers. As such
only measurement of 1-methylinosine was useful for providing a
predictive measure of leukemia in a patient.
16. The present invention comprises a methodology for the detection
of modified nucleosides in a biological or bodily fluid of a
patient suspected of having a tumor or other biologiclal disorder
in which an increased level of modified nucleosides is a result.
Suitable biological or bodily fluids may include but are not
limited to saliva, urine, blood, and cerebrospinal fluid. Preferred
modified nucleosides for detection in an assay provided herein are
psueouridine (.psi.), 7-methylinosine (m.sup.7I), and
1-methyladenosine (m.sup.1A). By detecting the levels of modified
nucleosides such as these in bodily fluid of a patient, the
presence of a tumor such as a glioma or meningioma in that patient
may be detected. In addition, the present invention may be utilized
to predict the responsiveness of a patient to a particular
chemotherapeutic regimen. For instance, a patient having a high
level of m.sup.1A may be predicted to respond less well to a
particular chemotherapeutic regimen as compared to a patient having
a high level of .psi.. As such, the treatment the patient receives
many modified to optimize the results of administration of certain
drugs or other compounds.
17. In one embodiment of the present invention, an in vitro assay
for the identification of compounds having the ability to decrease
modified nucleoside excretion from tumor cells. Compounds
identified in such an assay may then be tested in an in vivo model
as shown below or administered to a patient for treatment of a
disease such as cancer. A tumor cell line, either obtained from a
commercial source such as the ATCC (Rockville, Md.), or tumor cells
obtained from an excised human tumor are seeded onto a plate, such
as a 6-, 12-, 24-, 48- or 96-well plate at a sufficient
concentration to allow for normal cell growth in normal media. The
cells of certain wells are then treated with various concentrations
of a compound that may affect modified nucleoside production by the
cells. Such a compound may include dibutyrate (available from Sigma
Chemicals, St. Louis, Mo.) to which the cells may be exposed at
10.sup.-10 to 10.sup.-1 M concentrations. Modified nucleoside
levels are then characterized using procedures reported by Gehrke
et al. (J. Chromatography, 150: 455-476, 1978). An affinity column
may used as an initial step to partially purify the ribonucleosides
but any technique known by one skilled in the art for performing
such purifications may be utilized. Modified nucleosides contained
in this mixture may then analyzed using a methodology such as
reversed-phase high performance liquid chromatography (RP-HPLC).
Modified nucleosides may then be identified by retention time using
known standards and quantified by peak area integration. A compound
is determined to affect modified nucleoside production by the cells
if an increase or a decrease of the level of a particular modified
nucleoside is detected following exposure of the cells to the
compound. This information may then be extrapolated to determine
the in vivo levels needed to achieve a similar modification of
modified nucleoside production in a patient following dibutyrate
administration in vivo.
18. One obstacle to the design of effective therapies for treating
cancer is the variability in tumor cell reponses between
individuals. The assays provided herein may be utilized to
specifically characterize a patient's own tumor cells with respect
to the affect of compounds on modified nucleoside production. To
determine the responsiveness of an individual's tumor cells to a
particular treatment regimen, which may include chemotherapy,
radiation therapy, immunotherapy or other anti-cancer treatment, a
tumor may be resected from a patient and prepared as a primary
tumor cell suspension. Isolation and preparation of the tumor cells
may be performed using any of the procedures known to one skilled
in the art (see for example, Culture of Animal Cells: A Manual of
Basic Technique, 2.sup.nd Ed. (R. I. Freshney. 1987. Liss, Inc. New
York, N.Y.)). The cells may then be plated in culture dishes such
as a 96-well plates at a suitable concentration (preferably
10.sup.3 to 5.times.10.sup.6 cells/well) in normal media and
treated with various concentrations of a compound that may affect
modified nucleoside production by the cells. Modified nucleosides
levels may then be determined as described herein. An increase or
decrease in modified nucleoside levels following exposure of a
patient's cells to a chemotherapeutic compound or other treatment
may reflect the ability of that treatment to provide the patient
with a more favorable prognosis.
19. This system may also be tested using an animal model wherein
individual animals are inoculated with tumor cells (preferably
10.sup.2-10.sup.7 tumor cells). The animal may comprise any animal
suitable to laboratory testing, but is preferably a rat or a mouse.
A nu/nu mouse ("nude mouse") may be utilized where the tumor to be
tested is derived from a human. The tumor cells may have been
derived from any cancer, but preferably one of the brain. The level
of modified nucleoside in the bodily fluid of each animal is
determined prior to inoculation. The animal is then inoculate with
tumor cells and after a sufficient period of time during which the
tumor may proliferate, preferably 5-20 days, biological fluid is
again collected. The collected biological fluid is then tested for
the levels of modified nucleosides. The animals may be treated by a
treatment regimen either prior to inoculation or after inoculation
with the tumor cells. The modified nucleoside concentration of the
biological fluid is then determined and compared to the levels
detected in untreated mice. Both sets of data may also be compared
to the levels detected in healthy controls that have not been
inoculated with any tumor cells. The levels of modified nucleoside
detected in the biological fluid of each rat may also be correlated
to tumor size and overall health of the animal. If the amount of
modified nucleoside has decreased, this indicates that the
particular chemotherapeutic compound is effective for treating the
particular tumor type. If the levels of modified nucleoside has
increased, this indicates that chemotherapeutic compound is
ineffective for treating the particular tumor type. In this manner,
the effectiveness of a particular treatment regimen against a tumor
type can be ascertained.
20. It is useful in conducting such assays to have an antibody
having specificity to the modified nucleosides to be detected.
Polyclonal or monoclonal antibodies may be produced that have
specificity for psueouridine (.psi.), 7-methylinosine (m.sup.7I),
and 1-methyladenosine (m.sup.1A). The generation of antibodies to
1-methylinosine is described in U.S. Pat. No. 4,687,733. A similar
method may be utilized to generate antibodies to psueouridine
(.psi.), 7-methylinosine (m.sup.7I), and 1-methyladenosine
(m.sup.1A) by substituting one of these modified nucleosides for
1-methylinosine. Immunizations such as these are well known and the
reagents and methodologies for such immunizations are widely
available to one skilled in the art.
21. The present invention provides an assay for detection of
modified nucleosides in a biological sample such as bodily fluid.
Several techniques are available to one skilled in the art for the
detection of a quantity of antigen or antibody in a biological
fluid. Radioimmunoassays may be utilized as described by Levine, et
al. (J. Natl. Cancer Inst. 54: 341-343, 1984), Vold, et al. (Nuc.
Acids Res., 7: 193-204, 1979 and Cancer Res., 42: 5265-5269, 1982),
or Woodsworth, et al. (Bioch. Biophys. Res., 11+: 791-796, 1983),
for example. Radioimmunoassay may be carried out as described by
Farr, et al. (J. Infect. Dis. 103: 239-262, 1958) with certain
modifications as described in U.S. Pat. No. 4,687,733 (and
substituting .psi., m.sup.7I, or m.sup.1A for 1-methylinosine). The
affinity constants can be determined as described for the
antibodies as described by Muller, et al. (J. Immunol. Meth. 34:
345-352, 1980).
22. Other such methods include variations of the competitive
binding assay and the sandwich immunoassay. In a competitive assay,
antibody bound to a solid surface is contacted with a biological
sample containing an unknown antigen, and with a known quantity of
antigen complexed with a detectable label. In a sandwich assay,
antibody for the unknown antigen is bound to a solid surface, the
antigen is reacted with the antibody, and the amount of bound
antibody is measured by detection of the antigen with a second
antibody bound to a detectable label. A detectable label may be a
radioactive label such as .sup.125I, a fluorometric label such as
FITC or a colorimetric assay such as an enzyme label that causes
conversion of a substrate to a colored form.
23. In a typical competitive assay, such as that described by
Engvall et al. (Biochem. Biophys. Acta, 251: 427-434), the amount
of antigen is determined using an enzyme-labeled antibody and an
antibody coated tube. The labeled antibody bound to the tube is
contacted with a biological sample suspected of containing an
antigen, and with a known quantity of antigen, thus forming an
immunological complex. Following a wash step, the immunological
complex is contacted with a substrate solution that changes color
in the presence of the enzyme label on the antigen. The color
change of the substrate is determined by measuring the absorbance
of the solution at a particular wavelength. An ELISA may also be
used to detect a modified nucleoside.
24. Solid phase immunoassay devices may also be utilized in
practicing the present invention. A suitable solid phase
immunoassay device comprises a member of a ligand-receptor pair,
generally an antibody, hapten or antigen is bound to a solid
support. U.S. Pat. No. 4,366,241 describes a migration type assay
in which a membrane comprises, bound to it, the reagents needed to
perform the assay and further comprises an analyte detection zone
for identification of a target molecule. U.S. Pat. No. 4,770,853
describe other migration type devices comprising reagents attached
to colored labels for direct visual detection of the assay results
in the absence of additional reagents. U.S. Pat. No. 4,632,901
illustrates a flow-through type immunoassay device comprising
antibody bound to a porous membrane or filter to which is added a
liquid sample. Target analyte binds to the antibody as the liquid
flows through the membrane. Lableled antibody is then added, which
allows for visual detection of labeled antibody. U.S. Pat. Nos.
4,313,734, and 4,786,589 and 5,656,448 describe dipstick devices
useful for detection antigens from a body fluid such as urine.
25. The present invention may be practiced using any of such
detection devices. For instance, a dipstick comprising an antibody
having specificity to a modified nucleoside bound to solid support
may be inserted into a test solution comprising a biological fluid
such as bodily fluid into which a labeled anti-modified nucleoside
antibody has been added. The antibody bound to the dipstick and the
labeled antibody are both specific for a modified nucleoside, but
are specific to different epitopes. Binding of each antibody to the
modified nucleoside is not mutually exclusive with respect to one
another. As such, the antibody of the dipstick and the labeled
antibody may bind to the modified nucleoside to form a
sandwich.
26. To carry out this method, the dipstick, biological fluid and
labeled antibody are kept in contact with one another for a
sufficient period of time to allow for binding. The modified
nucleoside contained within the biological test sample is captured
as a complex comprising a solid support bound to an unlabelled
antibody bound to a modified nucleoside, he modified nucleoside
being further bound to a labeled antibody. The complex is then
processed to allow detection of the labeled antibody, allowing for
detection of the complex. Quantitation of the complex bound to the
dipstick is then accomplished by means known to one skilled in the
art. Comparison of the amount of bound modified nucleoside in a
test sample is then compared to a control sample (i.e., from a
tumor free patient). A greater amount of bound complex in the test
sample indicates a higher level of modified nucleoside in the test
biological sample.
27. The following non-limiting examples illustrate the inventors'
preferred methods for isolating a nucleic acid molecule having the
above-described properties. The following examples are provided for
illustrative purposes only and are not intended, nor should they be
construed, as limiting the invention in any manner. Those skilled
in the art will appreciate that modifications and variations of the
following examples can be made without exceeding the spirit or
scope of the present invention and claims.
EXAMPLE 1
Detection of MN in Patients having Brain Cancer
28. Using RP-HPLC, the concentration of three MNs (pseudouridine,
7-methylinosine, and 1-methyladenosine) in the urine of patients
diagnosed with gliomas (Glm) and meningiomas (Mng) was measured and
compared that detected in non-tumor craniotomy controls (NTC) and
healthy controls (HC).
29. To perform the assay, urine was collected pre-operatively from
three groups of subjects: tumor (n=151), non-tumor craniotomy (CC;
n=46) and HC (n=20) and stored at -20.degree. C. Each subject's
age, sex, tumor volume, mediaction, follow-up health and survival
rate was recorded. The tumor group was subdivided into three
groups: glioblastoma multiforme (GBM; n=52), medium grade glioma
(MG, n=47) and meningioma (MEN, n=52). Modified nucleosides were
characterized using procedures reported by Gehrke et al. (J.
Chromatography, 150: 455-476, 1978). A phenyl-boronate affinity
column was used as an initial step to partially purify the
ribonucleosides. Modified nucleosides contained in this mixture
were then analyzed by reversed-phase high performance liquid
chromatography (RP-HPLC) using a 4.times.300 mm .mu.Bondapak column
(waters Milford, Mass.) under the following conditions: buffer, 1%
methanol in0.01M sodium phosphate, pH 5.1; temperature, 25.degree.
C.; flow rate, 1 ml/min. Modified nucleosides were identified by
retention time using known standards and quantified by peak area
integration. These values were then normalized according to each
patient's creatine concentration (Sigma Diagnostics Creatinine Kit;
Sigma Chemicals, St. Louis, Mo.).
30. Pseudouridine and 1-methyladenosine levels were significantly
elevated in both Glm and Mng as compared to HC (FIG. 1). FIG. 1A
depicts modified nucleoside levels as means .+-.SEM and FIG. 1B
demonstrates the range of the MN values. The mean pseudourindine
levels are elevated in the glioblastoma (GBN, n=52) and meningioma
(MEN, n=52) tumor groups as compared to the healthy volunteer (HC,
n=20) and non-tumor craniotomy (CC, n=46) controls. The mean
1-methyladenosine levels are elevated in all three tumor groups in
the CC group compared to the HC group. However, due to the broad
range of levels observed in all groups, elevations of
1-methyladenosine were not statistically significant.
31. 7-methylinosine was significantly elevated in Mngs as compared
to NTC. 7-methylinosine and 1-methyladenosine were both elevated in
high-grade gliomas (n=28) as compared to medium-grade gliomas
(III-IV, n=14). In the Mng tumor group, 7-methylinosine was
significantly elevated in females (n=14) as compared to males
(n=9). Interestingly, a correlation between the tumor volumes and
the levels of any of the MNs was not found in any of the tumor
groups.
32. No differences in pseudouridine or 1-methyladenosine were found
with respect to gender or age in Glm, Mng or controls, but
7-methylinosine levels were found to decrease with increasing
age.
33. These results suggest that the characterization and
quantification of urinary MNs is useful in the diagnosis and
treatment of brain tumors. Furthermore, the data support the
hypothesis that it is altered tumor RNA metabolism and not tumor
cell proliferation that is responsible for the elevations of MNs in
brain tumor patients.
EXAMPLE 2
Differentiation of ""Poor Responders" and "Good Responders" by
Measurement of 1-Methyladenosine Levels
34. The pseudouridine, 7-methylinosine and 1-methyladenosine levels
of the glioblastoma patient group analyzed in Example 1 were
divided into two subgroups: high excretors (those patients having
levels above one standard deviation above the mean of the healthy
control ("HC") group) and low excretors (those patients having
levels below one standard deviation below the mean of the HC
group). The values were then plotted against patients' survival
from the time of treatment. The patients of the 1-methladenosine
high excretor giloblastoma (GBM) group (n=31) were found to have
significantly lower survival rates (Wilcoxon two-tail p=0.0167)
than the low excretors (n=23). The patients of the
1-methyladenosine high excretor midgrade glioma (M-G) group (n=36)
were also found to have significantly lower survival rates
(Wilcoxon two-tail p=0.037) than the low excretor group (n=21).
35. The data indicates a correlation between the levels of
1-methyladenosine excretion and prognosis. A patient having a high
excretor profile is predicted to have a worse prognosis than a
patient having a low excretor profile.
EXAMPLE 3
In vitro Assay for Compounds Capable of Reducing Modified
Nucleoside Production
36. The data suggests that compounds capable of slowing or
eliminating modified nucleoside production may be useful for
treating a disease such as cancer. The above-described data
indicates a correlation between the excretion of a high level of
modified nucleosides and poor prognosis. The present invention
provides an in vitro assay for the identification of compounds
hating the ability to decrease modified nucleoside excretion from
tumor cells. Compounds identified in such an assay may then be
tested in an in vivo model as shown below or administered to a
patient for treatment of a disease such as cancer.
37. Human glioma U373 MG cells are seeded onto a 96-well plate at
approximately 10.sup.5 cells/well in normal media and treated with
various concentrations of a compound that may affect modified
nucleoside production by the cells. Such a compound may include
dibutyrate (available from Sigma Chemicals, St. Louis, Mo.) to
which the cells may be exposed at 10.sup.-10 to 10.sup.-1 M
concentrations. Modified nucleoside levels are then characterized
using procedures reported by Gehrke et al. (J. Chromatography, 150:
455-476, 1978). A phenyl-boronate affinity column was used as an
initial step to partially purify the ribonucleosides. Modified
nucleosides contained in this mixture are then analyzed by
reversed-phase high performance liquid chromatography (RP-HPLC)
using a 4.times.300 mm .mu.Bondapak column (waters, Milford, Mass.)
under the following conditions: buffer, 1% methanol in 0.01M sodium
phosphate, pH 5.1; temperature, 25.degree. C.; flow rate, 1 ml/min.
Modified nucleosides are identified by retention time using known
standards and quantified by peak area integration. A compound is
determined to affect modified nucleoside production by the cells if
an increase or a decrease of the level of a particular modified
nucleoside is detected following exposure of the cells to the
compound.
38. The levels of the various nucleosides released by the cells in
the absence or presence of dibutyrate at various concentrations is
determined. This information may then be extrapolated to determine
the in vivo levels needed to achieve a similar modification of
modified nucleoside production in a patient following dibutyrate
administration in vivo.
EXAMPLE 4
In vitro Assay for Compounds Capable of Reducing Modified
Nucleoside Production by Autologous Tumor Cells
39. Variability between tumor types and between similar tumors in
different individuals has been a major obstacle in designing
effective therapies. The assay of the present invention may be
utilized to specifically characterize a patient's own tumor cells
with respect to the affect of compounds on modified nucleoside
production by tumor cells removed from a patient. In such a manner,
a therapeutic regimen may be specifically tailored to a particular
patient's tumor.
40. Following tumor resection from a patient, the tumor cells are
isolated using procedures well known and widely available to one
skilled in the art for preparing primary cells for culture.
Extratumoral cells and material are carefully removed from the
tumor fragment to reduce the likelihood of non-tumor cell
contamination The tumor is minced and the fragments treated with a
protease such as trypsin for a period of time sufficient to
generate a suspension containing single cells. The suspension is
then allowed to stand for a period of time sufficient to allow
large fragments to settle. The cells are then isolated and washed
in a suitable buffer such as phosphate-buffered saline (PBS). The
cells are then plated in 96-well plates at approximately 10.sup.5
cells/well in normal media and treated with various concentrations
of a compound that may affect modified nucleoside production by the
cells. Such a compound may include dibutyrate (available from Sigma
Chemicals. St. Louis, Mo.) to which the cells may be exposed at
10.sup.-10 to 10.sup.-1 M concentrations.
41. Modified nucleosides are characterized using procedures
reported by Gehrke et al. (J. Chromatography, 150: 455-476, 1978).
A phenyl-boronate affinity column was used as an initial step to
partially purify the ribonucleosides. Modified nucleosides
contained in this mixture were then analyzed by reversed-phase high
performance liquid chromatography (RP-HPLC) using a 4.times.300 mm
.mu.Bondapak column (waters, Milford, Mass.) under the following
conditions: buffer, 1% methanol in 0.01M sodium phosphate, pH 5.1;
temperature, 25.degree. C.; flow rate. 1 ml/min. Modified
nucleosides were identified by retention time using known standards
and quantified by peak area integration. These values were then
normalized according to each patient's creating concentration
(Sigma Diagnostics Creatinine Kit: Sigma Chemicals, St. Louis,
Mo.).
42. Those compounds having the ability to alter the patient's tumor
cell modified nucleoside production patterns are then considered
for administration to the patient. In such a manner, certain
compounds having the ability to alter autologous tumor cells of a
patient may be effectively utilized to treat the patient and
prevent tumor recurrence.
EXAMPLE 5
In vivo Model for Detecting Compounds having the Ability to Alter
Tumor Cell Modified Nucleoside Excretion
43. To determine the effectiveness of a chemotherapeutic regimen in
treating a particular type of cancer, the concentration of three
modified nucleosides (pseudouridine, 7-methylinosine, and
1-methyladenosine) in the urine of multiple rats is measured prior
to innoculation with tumor cells. Urine is collected from the
patient prior to treatment with a specific chemotherapeutic,
radioactive or immunological treatment protocol. The urine is
stored at -20.degree. C. Modified nucleosides are characterized
using procedures reported by Gehrke et al. (J. Chromatography, 150:
455-476, 1978). A phenyl-boronate affinity column is used as an
initial step to partially purify the ribonucleosides. Modified
nucleosides contained in this mixture are then analyzed by
reversed-phase high performance liquid chromatography (RP-HPLC)
using a 4.times.300 mm .mu.Bondapak column (waters, Milford, Mass.)
under the following conditions: buffer, 1% methanol in 0.01M sodium
phosphate, pH 5.1; temperature, 25.degree. C.; flow rate, 1 ml/min.
Modified nucleosides are identified by retention time using known
standards and quantified by peak area integration. These values are
then normalized according to each animal's creatine concentration
(Sigma Diagnostics Creatinine Kit; Sigma Chemicals, St. Louis,
Mo.).
44. Individual rats are then independently inoculated with
10.sup.6, 5.times.10.sup.6, or 10.sup.7 tumor cells. One week
later, urine is collected from each rat and control rats that have
not been inoculated with tumor cells. The concentration of
pseudouridine, 7-methylinosine, and 1-methyladenosine is determined
and compared to the pre-inoculation levels. Both sets of data are
further compared to the levels detected in healthy controls that
have not been inoculated with any tumor cells. Treatment with a
chemotherapeutic compound is then initiated in certain rats (the
"treated" group). Treatment is with a chemotherapeutic compound
believed to be effective against the particular tumor type from
which the tumor cells were derived.
45. Urine is collected from each rat (treated and untreated) at
seven day intervals and assayed for compared to the previous
assays. One month after treatment with the compound has begun,
urine is once again collected and assayed for the concentration of
pseudouridine, 7-methylinosine, and 1-methyladenosine. The levels
of modified nucleoside detected in the urine of each rat is then
correlated to tumor size and overall health of the animal. If the
amount of modified nucleoside has decreased, this indicates that
the particular chemotherapeutic compound is effective for treating
the particular tumor type. If the levels of modified nucleoside has
increased, this indicates that chemotherapeutic compound is
ineffective for treating the particular tumor type. In this manner,
the effectiveness of a particular chemotherapeutic regimen against
a tumor type can be ascertained.
EXAMPLE 6
Kit for Detection of Modified Nucleosides in a Biological Fluid of
a Patient
46. A kit for detecting modified nucleosides in a biological fluid
of a patient is provided herein. The kit comprises a dipstick
comprising a solid surface comprising three zones to which are
separately bound a monoclonal antibody to psueouridine (.psi.),
7-methylinosine (m.sup.7I), or 1-methyladenosine (m.sup.1A). The
dipstick is placed into a container comprising urine from either a
patient suspected of having a brain tumor or a control sample
(i.e., from a patient not suspected of having a tumor) and a
labeled antibody specific to psueouridine (.psi.), 7-methylinosine
(m.sup.7I), or 1-methyladenosine (m.sup.1A). The labeled antibody
is conjugated to alkaline phosphatase (specific activity>1400
U/mg) at a 1:1 molar ratio using the one step glutaraldehyde method
followed by purification of the antibody conjugate on a
diethylaminoethyl Sephadex A50 (5 ml) column using a liner gradient
(0-1M sodium chloride). Conjugate samples (3.0 ml) are collected
and assayed for enzyme activity. The antibody is then diluted to a
working concentration.
47. The dipstick, urine and labeled antibody are exposed to one
another for approximately three minutes and inserted into a tube
containing substrate solution (5-bromo-4-chloro indoyl
phosphate-nitroblue tetrazolium) with shaking. The tube is allowed
to stand for three minutes and removed from the tube. Each zone of
the dipstick that comprises bound antibody specific to specific to
psuecouridine (.psi.), 7-methylinosine (m.sup.7I), or
1-methyladenosine (m.sup.1A) is observed. The presence of a blue
color formation at each zone indicates that specific to
psueouridine (.psi.), 7-methylinosine (m.sup.7I), or
1-methyladenosine (m.sup.1A) is bound to the dipstick. The levels
of the different modified nucleosides in the experimental samples
are then compared to that of control samples. In general, an
increased or decreased amount of modified nucleoside is detected in
the experimental sample as compared to the control samples.
48. As disclosed herein, the present invention provides the
reagents and methodologies for the detection of modified
nucleosides for detection of cancer or other conditions wherein
increased levels of modified nucleosides results. While a preferred
form of the invention has been shown in the drawings and described,
since variations in the preferred form will be apparent to those
skilled in the art, the invention should not be construed as
limited to the specific form shown and described, but instead is as
set forth in the claims.
* * * * *