U.S. patent application number 11/861655 was filed with the patent office on 2008-06-05 for methods of using foxp3 levels to predict the outcome of organs undergoing acute rejection.
Invention is credited to Manikkam Suthanthiran.
Application Number | 20080131441 11/861655 |
Document ID | / |
Family ID | 39476057 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131441 |
Kind Code |
A1 |
Suthanthiran; Manikkam |
June 5, 2008 |
Methods of Using FOXP3 Levels to Predict the Outcome of Organs
Undergoing Acute Rejection
Abstract
A method for assessing risk of losing a transplanted organ by a
patient having an episode of acute rejection of the transplanted
organ is described. The method includes obtaining from the patient
a cell sample from the transplanted organ or peripheral blood,
determining a level of FOXP3 in the cell sample, and correlating
the level with the risk of loss of the transplanted organ, wherein,
compared to a control level, a significantly greater level of FOXP3
in the cell sample from the transplanted organ or a significantly
lower level of FOXP3 in the cell sample from the peripheral blood
correlates with a decreased risk of loss of the transplanted
organ.
Inventors: |
Suthanthiran; Manikkam;
(Scarsdale, NY) |
Correspondence
Address: |
HOFFMANN & BARON, LLP
6900 JERICHO TURNPIKE
SYOSSET
NY
11791
US
|
Family ID: |
39476057 |
Appl. No.: |
11/861655 |
Filed: |
September 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60848040 |
Sep 26, 2006 |
|
|
|
Current U.S.
Class: |
424/144.1 ;
424/130.1; 435/29; 435/6.16; 514/178 |
Current CPC
Class: |
C12Q 2600/106 20130101;
A61K 35/14 20130101; C07K 16/2809 20130101; A61K 31/56 20130101;
C07K 16/28 20130101; G01N 2800/50 20130101; G01N 2333/4703
20130101; G01N 2800/52 20130101; G01N 33/6872 20130101; G01N
33/6893 20130101; A61K 2039/505 20130101; A61P 43/00 20180101; C12Q
2600/158 20130101; G01N 2800/245 20130101; C12Q 1/6876 20130101;
C12Q 2600/16 20130101 |
Class at
Publication: |
424/144.1 ;
435/29; 435/6; 514/178; 424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/02 20060101 C12Q001/02; C12Q 1/68 20060101
C12Q001/68; A61P 43/00 20060101 A61P043/00; A61K 31/56 20060101
A61K031/56 |
Goverment Interests
[0002] The invention described in this application was made with
funds from the National Institutes of Health, Grant Numbers RO1
A151652 and AI60706. The United States government has certain
rights in the invention.
Claims
1. A method for assessing risk of losing a transplanted organ by a
patient having an episode of acute rejection of the transplanted
organ, the method comprising: (a) obtaining from the patient a cell
sample from the transplanted organ or from peripheral blood; (b)
determining a level of FOXP3 in the cell sample; and (c)
correlating the level with the risk of loss of the transplanted
organ, wherein, compared to a control level, a significantly
greater level of FOXP3 in the cell sample from the transplanted
organ or a significantly lower level of FOXP3 in the cell sample
from the peripheral blood correlates with a decreased risk of loss
of the transplanted organ, or a level of FOXP3 that is not
significantly greater in the cell sample from the transplanted
organ or a level of FOXP3 that is not significantly lower in the
cell sample from peripheral blood correlates with an increased risk
of loss of the transplanted organ.
2. A method according to claim 1, wherein the level of FOXP3 is
determined by measuring the level of FOXP3 in RNA.
3. A method according to claim 1, wherein the level of FOXP3 is
determined by measuring the level of FOXP3 protein.
4. A method according to claim 1, wherein the method further
comprises assessing the likelihood of the patient's responding to
anti-rejection treatment, wherein, compared to a control level, a
significantly greater level of FOXP3 in the cell sample from the
transplanted organ, or a significantly lower level of FOXP3 in the
cell sample from the peripheral blood correlates with an increased
likelihood of responding to anti-rejection treatment, and a level
of FOXP3 that is not significantly greater in the cell sample from
the transplanted organ or a level of FOXP3 that is not
significantly lower in the cell sample from peripheral blood
correlates with a decreased likelihood of responding to
anti-rejection treatment.
5. A method according to claim 5, wherein the transplanted organ is
a kidney.
6. A method according to claim 3, wherein the cell sample from the
kidney is urine.
7. A method according to claim 1, wherein the transplanted organ is
a heart.
8. A method according to claim 1, wherein the transplanted organ is
a liver.
9. A method according to claim 1, wherein the transplanted organ is
a lung.
10. A method according to claim 1, wherein the transplanted organ
is a pancreas.
11. A method according to claim 1, wherein the transplanted organ
is a pancreatic islets.
12. A method according to claim 1, wherein the transplanted organ
is intestine.
13. A method according to claim 1, further comprising informing the
patient whether the patient is at decreased or increased risk of
loss of the transplanted organ.
14. A method according to claim 1, further comprising prescribing
to the patient at increased risk of loss of the transplanted organ
a treatment to prevent loss of the transplanted organ.
15. A method according to claim 14, wherein the treatment comprises
an effective amount of a pharmaceutical composition to prevent loss
of the transplanted organ.
16. A method according to claim 12, wherein the pharmaceutical
composition comprises a steroid pulse.
17. A method according to claim 13, wherein the steroid is a
corticosteroid.
18. A method according to claim 12, wherein the pharmaceutical
composition comprises an antibody.
19. A method according to claim 14, wherein the antibody is
thymoglobin or OKT3.
20. A method according to claim 14, wherein the treatment comprises
plasmapheresis.
21. A method according to claim 5, further comprising determining
the patient's serum creatinine level in peripheral blood, and
correlating the level with the risk of loss of the transplanted
kidney, wherein, compared to a control level, a significantly
greater level of serum creatinine in peripheral blood correlates
with an increased risk of loss of the transplanted kidney, or a
level of serum creatinine in peripheral blood that is not
significantly greater correlates with a decreased risk of loss of
the transplanted kidney.
22. A method for assessing likelihood of responding to
anti-rejection treatment by a patient having an episode of acute
rejection of the transplanted organ, the method comprising: (a)
obtaining from the patient a cell sample from the transplanted
organ or from peripheral blood; (b) determining a level of FOXP3 in
the cell sample; and (c) correlating the level with the likelihood
of responding to anti-rejection treatment; wherein, compared to a
control level, a significantly greater level of FOXP3 in the cell
sample from the transplanted organ or a significantly lower level
of FOXP3 in the cell sample from the peripheral blood correlates
with an increased likelihood of responding to anti-rejection
treatment, or a level of FOXP3 that is not significantly greater in
the cell sample from the transplanted organ or a level of FOXP3
that is not significantly lower in the cell sample from peripheral
blood correlates with a decreased likelihood of responding to
anti-rejection treatment.
23. A method for assessing the likelihood of reversing an acute
rejection of a transplanted organ by a patient having an episode of
acute rejection of the transplanted organ, the method comprising:
(a) obtaining from the patient a cell sample from the transplanted
organ or from peripheral blood; (b) determining a level of FOXP3 in
the cell sample; (c) correlating the level with the likelihood of
reversing acute rejection of the transplanted organ; wherein,
compared to a control level, a significantly greater level of FOXP3
in the cell sample from the transplanted organ or a significantly
lower level of FOXP3 in the cell sample from the peripheral blood
correlates with an increased likelihood of reversing acute
rejection of the transplanted organ, or a level of FOXP3 that is
not significantly greater in the cell sample from the transplanted
organ or a level of FOXP3 that is not significantly lower in the
cell sample from peripheral blood correlates with a decreased
likelihood of reversing acute rejection of the transplanted organ.
Description
[0001] This application asserts priority to U.S. Provisional
Application Ser. No. 60/848,040 filed on Sep. 26, 2006, the
specification of which is hereby incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Acute rejection of an organ, transplanted from one human to
another, is an important risk factor for allograft failure. The
outcome of acute rejection is, however, difficult to predict.
[0004] Currently, observation of histologic features in allograft
tissue obtained by core needle biopsy is the best predictor whether
an acute rejection will respond to anti-rejection therapy. However,
the invasive procedure of allograft biopsy is associated with
complications such as bleeding, arteriovenous fistula, and even
graft loss. Thus, there is a need for a non-invasive method for
determining whether a patient suffering from acute rejection of a
transplant organ is at risk of loss of the transplanted organ.
SUMMARY OF THE INVENTION
[0005] The above need has been met by the present invention, which
provides in one embodiment a method for assessing risk of losing a
transplanted organ by a patient having an episode of acute
rejection of the transplanted organ, the method comprising
obtaining from the patient a cell sample from the transplanted
organ or from peripheral blood, determining a level of FOXP3 in the
cell sample, and correlating the level with the risk of loss of the
transplanted organ, wherein, compared to a control level, a
significantly greater level of FOXP3 in the cell sample from the
transplanted organ or a significantly lower level of FOXP3 in the
cell sample from the peripheral blood correlates with a decreased
risk of loss of the transplanted organ, or a level of FOXP3 that is
not significantly greater in the cell sample from the transplanted
organ or a level of FOXP3 that is not significantly lower in the
cell sample from peripheral blood correlates with an increased risk
of loss of the transplanted organ.
[0006] In another embodiment of the present invention, a method for
assessing likelihood of responding to anti-rejection treatment by a
patient having an episode of acute rejection of the transplanted
organ, the method comprises obtaining from the patient a cell
sample from the transplanted organ or from peripheral blood,
determining a level of FOXP3 in the cell sample; and correlating
the level with the likelihood of responding to anti-rejection
treatment wherein, compared to a control level, a significantly
greater level of FOXP3 in the cell sample from the transplanted
organ or a significantly lower level of FOXP3 in the cell sample
from the peripheral blood correlates with an increased likelihood
of responding to anti-rejection treatment, or a level of FOXP3 that
is not significantly greater in the cell sample from the
transplanted organ or a level of FOXP3 that is not significantly
lower in the cell sample from peripheral blood correlates with a
decreased likelihood of responding to anti-rejection treatment.
[0007] In another embodiment of the present invention, a method for
assessing the likelihood of reversing an acute rejection of a
transplanted organ by a patient having an episode of acute
rejection of the transplanted organ, the method comprises obtaining
from the patient a cell sample from the transplanted organ or from
peripheral blood, determining a level of FOXP3 in the cell sample,
correlating the level with the likelihood of reversing acute
rejection of the transplanted organ, wherein, compared to a control
level, a significantly greater level of FOXP3 in the cell sample
from the transplanted organ or a significantly lower level of FOXP3
in the cell sample from the peripheral blood correlates with an
increased likelihood of reversing acute rejection of the
transplanted organ, or a level of FOXP3 that is not significantly
greater in the cell sample from the transplanted organ or a level
of FOXP3 that is not significantly lower in the cell sample from
peripheral blood correlates with a decreased likelihood of
reversing acute rejection of the transplanted organ.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1. Level of FOX3 mRNA in urinary cells. Box plots show
the 10th, 25.sup.th, 50th (median), 75.sup.th, and 90th percentile
values for log-transformed ratios of mRNA copies to 18S rRNA copies
for FOX3, CD25, CD3.epsilon., and perforin in urine samples
obtained from 36 subjects with biopsy-confined acute rejection, 18
subjects with biopsy-confirmed chronic allograft nephropathy, and
29 subjects with stable graft function and normal biopsy results.
The levels of mRNA for FOX3, CD25, CD3.epsilon., and perforin were
higher in the urinary cells from subjects with acute rejection than
in the subjects with chronic allograft nephropathy or normal biopsy
results. P values are based on the Kruskal-Wallis test, with the
log-transformed mRNA levels treated as the dependent variable.
Dunn's multiple-comparison test showed that levels of FOXP3 mRNA in
the acute-rejection group were higher than those in both the group
with chronic allograft nephropathy (P<0.05) and the group with
normal biopsy results (P<0.01) (Panel A). CD25 mRNA levels were
higher in the acute-rejection group than in both the group with
chronic allograft nephropathy (P<0.001) and the group with
normal biopsy results (P<0.001) (Panel B). CD3.epsilon. mRNA
levels were higher in the acute-rejection group than in both the
group with chronic allograft nephropathy (P<0.01) and the group
with normal biopsy results (P<0.001); CD3.epsilon. mRNA levels
were also higher in the group with chronic allograft nephropathy
than in the group with normal biopsy results P<0.05) (Panel C).
Perforin mRNA levels were higher in the acute-rejection group than
in both the group with chronic allograft nephropathy (P<0.001)
and the group with normal biopsy results (P<0.001) (Panel D). In
all cases, log-transformed level, normalized for 18S rRNA, are
shown.
[0009] FIG. 2. Correlation between Levels of FOXP3 mRNA in Urinary
Cells and Reversal of an Episode of Acute Rejection. Box plots show
the 10th, 25th, 50th (median), 75th, and 90th percentiles for
levels of mRNA for FOXP3, CD25, CD3.epsilon., and perforin in urine
samples obtained from 26 subjects with successful reversal of acute
rejection (classified as reversible and defined by the return of
serum creatinine levels to within 15 percent of prerejection levels
within four weeks after the initiation of antirejection treatment)
and 10 patients without reversal of acute rejection
(nonreversible). The levels of mRNA for FOXP3 but not for CD25,
CD3.epsilon., and perforin were significantly higher in subjects
with reversible acute rejection than in subjects with nonreversible
acute rejection. Two-tailed P values are based on the Mann-Whitney
test. In all cases, log-transformed levels, normalized for 18S
rRNA, are shown.
[0010] FIG. 3. Receiver-Operating-Characteristic (ROC) Curves for
mRNA Levels. The fraction of true positive results (sensitivity)
and false positive results (1-specificity) for levels of mRNA for
FOXP3, CD25, CD3.epsilon., and perforin, each normalized for 18S
rRNA, as predictors of reversal of acute rejection are shown. The
calculated area under the curve was 0.85 (95 percent confidence
interval, 0.71 to 0.99) for FOXP3 mRNA levels (Panel A) but only
0.63 (95 percent confidence interval, 0.42 to 0.84) for CD25 mRNA
levels (Panel B), 0.60 (95 percent confidence interval, 0.39 to
0.81) for CD3.epsilon. mRNA levels (Panel C), and 0.58 (95 percent
confidence interval, 0.38 to 0.79) for perforin mRNA levels (Panel
D). A P value of 0.5 is no better than that expected by chance (the
null hypothesis), and a P value of 1.0 reflects a perfect
indicator. Of the four mRNA measures, only FOXP3 predicts
successful reversal significantly better than chance (P=0.001).
[0011] FIG. 4. Relative Risk (RR) of Graft Failure after an Episode
of Acute Rejection. The rates and relative risks of graft failure
within six months after an episode of acute rejection for thirds of
FOXP3, CD25, CD3.epsilon., and perforin mRNA levels are shown. The
graft-failure rate was 50 percent and the relative risk was 6 for
the lowest third of FOXP3 mRNA levels; 25 percent and 3,
respectively, for the middle third; and 8 percent for the highest
third (P=0.02 by the chi-square test for linear trend) (Panel A).
There was no relationship between graft failure after an episode of
acute rejection and the thirds of mRNA levels for CD25,
CD3.epsilon., and perforin (Panels B, C, and D).
[0012] FIG. 5. Levels of mRNA in Urinary Cells and the Time from
Kidney Transplantation to the Development of Acute Rejection. The
relationship between levels of mRNA in urine samples and the time
from kidney transplantation to the development of biopsy-confirmed
acute rejection is shown, along with Spearman's rank-order
correlation (r.sub.s). A significant inverse relationship between
the levels of FOXP3 mRNA and CD25 mRNA in urinary cells and the
time from kidney transplantation to the development of acute
rejection was found (Panels A and B). There was no such
relationship between levels of CD3.epsilon. mRNA or perforin mRNA
in urinary cells and the time from kidney transplantation to the
development of acute rejection (Panels C and D). Log-transformed
mRNA levels, normalized for 18S rRNA, are shown. The numbers of
days from renal transplantation to the development of acute
rejection are shown on a log scale.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The invention is based on the surprising discovery by the
inventor that the level of forkhead box P3 (FOXP3), measured during
an episode of acute rejection, in a cell sample from a transplanted
organ or from peripheral blood of a patient is useful for assessing
the outcome of acute rejection. It has also been discovered by the
inventor that, compared to a control level, a significantly greater
level of FOXP3 during an episode of acute rejection in the cell
sample from the transplanted organ or a significantly lower level
of FOXP3 during an episode of acute rejection in the cell sample
from the peripheral blood correlates with a decreased risk of loss
of the transplanted organ. Similarly, compared to a control level,
a level of FOXP3 during an episode of acute rejection that is not
significantly greater in the cell sample from the transplanted
organ or a level of FOXP3 during an episode of acute rejection that
is not significantly lower in the cell sample from peripheral blood
correlates with an increased risk of loss of the transplanted
organ.
[0014] The patient is any human having an episode of acute
rejection of a transplanted organ. Any organ suitable for
transplantation can be susceptible to an episode of acute
rejection. Examples of such organs include kidney, heart, liver,
lung, interstines, pancreas, pancreatic islets, etc.
[0015] An episode of acute rejection of an organ can be caused by
an antibody-mediated or cell-mediated immune response. The cells
involved in a cell-mediated immune response include, for example,
activated cytotoxic T cells. An episode of acute rejection
typically occurs within fourteen days, more typically within ten
days, and even more typically within five days after a transplant
if the patient is not taking an immunosuppressant drug.
[0016] However, most if not all transplant patients are given
immunosuppressant drugs. Thus, an episode of acute rejection
generally occurs within about one year of a transplanted organ,
more generally within about nine months, even more generally within
about six months, and most generally within about three months
after transplant of an organ. Acute rejection, however, can occur
at any time during the life of a transplanted organ. Further, a
patient can have more than one episode of acute rejection of a
transplanted organ.
[0017] In one aspect, the invention provides a method for assessing
risk of losing a transplanted organ by a patient suffering from
acute rejection of the transplanted organ.
[0018] In another aspect, the invention provides a method for
assessing likelihood of responding to anti-rejection treatment by a
patient suffering from acute rejection of a transplanted organ.
[0019] In another aspect, the invention provides a method for
assessing the likelihood of reversing an acute rejection of a
transplanted organ by a patient suffering from acute rejection of
the organ.
[0020] Each of the methods of the present invention can be used
alone, or in combination with one or more, or all, of the other
methods.
[0021] The first step in the methods of the present invention
comprises obtaining from the patient a cell sample from the
transplanted organ or from peripheral blood. Suitable methods for
obtaining a cell sample are provided below in the "General Methods"
section.
[0022] The second step in the methods comprises determining the
level of FOXP3 in the cell sample. The determination of the level
of FOXP3 in the cell sample can be made by any method known to
those skilled in the art. In one embodiment, the level of FOXP3
mRNA or the corresponding level of cDNA is determined. In another
embodiment, the level of FOXP3 protein is determined. Suitable
methods are provided below in the "General Methods" section.
Method for Accessing Risk of Loss of Transplanted Organ
[0023] In this aspect of the present invention, the first and
second steps of the method are described above. The next step in
the method comprises correlating the level of FOXP3 with the risk
of loss of the transplanted organ.
[0024] When compared to a control level, a significantly greater
level of FOXP3 in the cell sample from the transplanted organ or a
significantly lower level of FOXP3 in the cell sample from the
peripheral blood correlates with a decreased risk of loss of the
transplanted organ. The decreased risk varies in different
patients, and the type of organ transplanted. Generally, the
decreased risk is at least about 25%, at least about 50%, at least
about 75%, or at least about 90%.
[0025] When compared to a control level, a level of FOXP3 that is
not significantly greater in the cell sample from the transplanted
organ or a level of FOXP3 that is not significantly lower in the
cell sample from peripheral blood correlates with an increased risk
of loss of the transplanted organ. The increased risk varies in
different patients, and the organ transplanted. Generally, the
increased risk is at least about 25%, at least about 50%, at least
about 75%, or at least about 90%.
[0026] When the transplanted organ is a kidney, the method for
assessing risk of a patient suffering from acute rejection
optionally further comprises determining the patient's serum
creatinine protein level. The determination of the level of serum
creatinine can be made by any method known to those skilled in the
art. Suitable methods are provided below in the "General Methods"
section below.
[0027] The next step in this embodiment comprises correlating the
level of serum creatinine in peripheral blood with risk of loss of
the transplanted organ. A significantly greater level of serum
creatinine in peripheral blood correlates with an increased risk of
loss of the transplanted kidney. A level of serum creatinine in
peripheral blood that is not significantly greater correlates with
a decreased risk of loss of the transplanted kidney.
[0028] Generally, the level of serum creatinine in peripheral blood
is considered to be significantly greater if the level is at least
about 25% greater than the level of creatinine in a control
sample.
[0029] In this embodiment, a control sample is typically the level
of serum creatinine in peripheral blood of a healthy person or a
person with a well-functioning (e.g., stable) transplant. For
example, the normal level of serum creatinine in a healthy person
or a person with a well-functioning transplant is generally about
0.8-1.6 milligrams/deciliter. In either case, the person may be the
patient or a person different from the patient.
[0030] It is not necessary to determine the level of creatinine in
a control sample every time the method is conducted. For example,
the serum creatinine level from the patient can be compared to that
of one or more previously determined control samples or to a level
recognized by the physician or clinician conducting the method, or
by a consensus of medical and/or clinical practitioners.
[0031] In another embodiment, the method further comprises
informing the patient whether the patient is at decreased or
increased risk of loss of the transplanted organ. The information
that a patient is at risk of loss of a transplanted organ is
useful. Such patients can be prescribed and/or administered a
treatment to prevent loss of the transplanted organ.
[0032] In one embodiment, the treatment comprises administering to
the patient an effective amount of a pharmaceutical composition to
prevent loss of the transplanted organ. Such pharmaceutical
compositions are well known to those skilled in the art, and
include, for example a steroid pulse, an antibody, etc.
[0033] For example, a steroid pulse therapy can include the
administration for three to six days of a high dose corticosteroid
(e.g., greater than 100 mg). An example of an antibody therapy
includes the administration for seven to fourteen days of the
polyclonal antibody Thymoglobin or the monoclonal antibody,
OKT3.
[0034] Another example of a treatment that can be administered is
plasmapheresis. Plasmapheresis is a process in which the fluid part
of the blood (i.e., plasma) is removed from blood cells. Typically,
the plasma is removed by a device known as a cell separator. The
cells are generally returned to the person undergoing treatment,
while the plasma, which contains antibodies, is discarded.
Method for Assessing Likelihood of Responding to Anti-Rejection
Treatment
[0035] In this aspect of the present invention, the first and
second steps of the method are described above. The next step in
the method comprises correlating the level of FOXP3 with the
likelihood of the patient in responding to anti-rejection
treatment.
[0036] When compared to a control level, a significantly greater
level of FOXP3 in the cell sample from the transplanted organ or a
significantly lower level of FOXP3 in the cell sample from the
peripheral blood correlates with an increased likelihood of
responding to anti-rejection treatment. The increased likelihood
varies in different patients and the type of organ transplanted.
Generally, the increased likelihood of responding to anti-rejection
treatment means the likelihood is increased by at least about 25%,
more preferably by at least about 50%, and even more preferably by
at least about 75%. Optimally, the patient responds to
anti-rejection therapy such that any risk of complete failure of
the transplanted organ is completely eliminated.
[0037] When compared to a control level, a level of FOXP3 that is
not significantly greater in the cell sample from the transplanted
organ or a level of FOXP3 that is not significantly lower in the
cell sample from peripheral blood correlates with a decreased
likelihood of responding to anti-rejection treatment. The decreased
likelihood varies in different patients, and the type of organ
transplanted. Generally, the decreased likelihood of responding to
anti-rejection treatment means the likelihood is decreased by at
least about 25%, by at least about 50%, and even by at least about
75%.
[0038] Examples of anti-rejection treatments include those
treatment described above for preventing rejection of a
transplanted organ.
Method for Assessing Likelihood of Reversing Acute Rejection
[0039] In this aspect of the present invention, the first and
second steps of the method are described above. The next step in
the method comprises correlating the level of FOXP3 with the
likelihood of reversing acute rejection of the transplanted
organ.
[0040] When compared to a control level, a significantly greater
level of FOXP3 in the cell sample from the transplanted organ or a
significantly lower level of FOXP3 in the cell sample from the
peripheral blood correlates with an increased likelihood of
reversing acute rejection of the transplanted organ. The increased
likelihood varies in different patients and the type of organ
transplanted. Generally, the increased likelihood of reversing
acute rejection of the transplanted organ means that the likelihood
is increased by at least about 25%, more preferably by at least
about 50%, and even more preferably by at least about 75%.
Optimally, rejection of the transplanted organ is completely
eliminated.
[0041] When compared to a control level, a level of FOXP3 that is
not significantly greater in the cell sample from the transplanted
organ or a level of FOXP3 that is not significantly lower in the
cell sample from peripheral blood correlates with a decreased
likelihood of reversing acute rejection of the transplanted organ.
The decreased likelihood varies in different patients and the type
of organ transplanted. Generally, the decreased likelihood of
reversing acute rejection means the likelihood is decreased by at
least about 25%, by at least about 50%, and even by at least about
75%.
General Methods
[0042] A cell sample from a transplanted organ or peripheral blood
can be obtained from a patient by any method known to those in the
art. Examples of such cell samples include transplant tissue
biopsy, blood, urine, bile, bronchoalveolar lavage fluid, and
pericardial fluid. Suitable methods include, for example, venous
puncture of a vein to obtain a blood sample and collection of a
urine specimen.
[0043] Any method known to those in the art can be employed for
determining the level of FOXP3 mRNA. Typically, total RNA, which
includes mRNA, is isolated. RNA can be isolated from the sample by
any method known to those in the art. For example, commercial kits,
such as the TRI Reagent.RTM. commercially available from Molecular
Research Center, Inc. (Cincinnati, Ohio), can be used to isolate
RNA.
[0044] The quantification of FOXP3 mRNA from total mRNA from the
biological sample can be performed by any method known to those in
the art. For example, kinetic, quantitative PCR involves reverse
transcribing FOXP3 mRNA by using reverse-transcriptase polymerase
chain reaction (RT-PCR) to obtain FOXP3 cDNA. The cDNA can then,
for example, be amplified by PCR followed by quantitation using a
suitable detection apparatus. See example 1 below for a description
of the quantitation of FOXP3 mRNA by kinetic, quantitative PCR.
[0045] Generally, the isolated FOXP3 mRNA may be amplified by
methods known in the art. Amplification systems utilizing, for
example, PCR or RT-PCR methodologies are known to those skilled in
the art. For a general overview of amplification technology, see,
for example, Dieffenbach et al., PCR Primer: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, New York (1995). For example,
levels of FOXP3 mRNA can be determined using kinetic, quantitative
PCR.
[0046] An alternative method for determining the level of FOXP3
mRNA includes the use of molecular beacons and other labeled probes
useful in, for example multiplex PCR. In a multiplex PCR assay, the
PCR mixture contains primers and probes directed to the FOXP3 PCR
product. Typically, a single fluorochrome is used in the assay. The
molecular beacon or probe is detected to determine the level of
FOXP3 mRNA. Molecular beacons are described, for example, by Tyagi
and Kramer (Nature Biotechnology 14, 303-308, 1996) and by Andrus
and Nichols in U.S. Patent Application Publication No.
20040053284.
[0047] Another method includes, for instance, quantifying cDNA
(obtained by reverse transcribing the FOXP3 mRNA) using a
fluorescence based real-time detection method, such as the ABI
PRISM 7700 or 7900 Sequence Detection System [TaqMan.RTM.]
commercially available from Applied Biosystems, Foster City, Calif.
or similar system as described by Heid et al., (Genome Res. 1996;
6:986-994) and Gibson et al. (Genome Res. 1996; 6:995-1001).
[0048] Any method known in the art can be used for determining the
protein level of FOXP3 in the cell sample from a transplanted organ
or peripheral blood, or serum creatinine levels from peripheral
blood.
[0049] Suitable methods for determining protein levels include an
ELISA and a standard blot. Briefly, these assays are normally based
on incubating an antibody specific to the protein with a sample
suspected of containing the protein, and detecting the presence of
a complex between the antibody and the protein.
[0050] Alternatively, commercial kits can be utilized. An example
of a commercial kit for determining creatinine level is the
QuantiChrom.TM. Creatinine Assay Kit from BioAssay Systems
(Hayward, Calif.).
[0051] Generally, the level of FOXP3 in a cell sample is
significantly greater if the gene expression of FOXP3 is
heightened. For example, a discriminatory level for heightened gene
expression (e.g., the baseline magnitude of gene expression) of
FOXP3 is defined as the mean.+-.95% confidence interval of a group
of values observed in nonrejecting transplants (e.g., control
values, i.e., control levels). The group of values as used herein
includes, for example, a minimum of at least about 2 values, more
preferably a minimum of at least about 10 values, most preferably a
minimum of at least about 20 values. The group of values as used
herein includes, for example, a maximum of at most about 500
values, more preferably a maximum of at most about 100 values, most
preferably a maximum of at most about 50 values.
[0052] Heightened gene expression of FOXP3 is considered to be
significantly greater if the value is greater than the mean .+-.95%
confidence interval of a group of values observed in nonrejecting
transplants. Similarly, the level of FOXP3 in the cell sample is
considered to be significantly lower if the FOXP3 value is lower
than the mean.+-.95% confidence interval of a group of values
observed in nonrejecting transplants.
[0053] The level of FOXP3 is typically considered not significantly
greater if the level of FOXP3 in a cell sample is not greater than
the mean.+-.95% confidence interval of a group of values observed
in nonrejecting transplants. The level of FOXP3 is normally
considered not significantly lower if the level in a cell sample is
not lower than the mean.+-.95% confidence interval of a group of
values observed in nonrejecting transplants.
[0054] Statistical analysis in the above mean.+-.95% confidence
interval of a group of values observed in nonrejecting transplants
was performed with a x.sup.2 test.
[0055] In another embodiment, the level of FOXP3 in a cell sample
is significantly greater if the log-transformed mean (.+-.SE) ratio
of FOXP3 mRNA copies to 18S-rRNA copies is higher relative to a
control ratio in nonrejecting organs, as determined by the
Kruskal-Wallis test. For example, a significantly greater ratio is
typically at least about .+-.SE 3.0, more typically between .+-.SE
3.0 and 5.0, and most typically between .+-.SE 3.8 and 4.7.
[0056] Similarly, the level of FOXP3 in a cell sample is
significantly lower if the log-transformed mean (.+-.SE) ratio of
FOXP3 mRNA copies to 18S-rRNA copies is reduced relative to a
control ratio (i.e., control values, control levels) in
nonrejecting organs, as determined by the Kruskal-Wallis test. For
example, a typical nonrejection organ control ratio is not more
than about 2.5, more typically 1.0 to 2.5, and most typically from
1.3 and 2.0.
[0057] In yet another embodiment, the control values (control
levels) observed in nonrejecting organs, e.g., kidneys, may be
defined as the level of FOXP3 of the same patient before the organ
transplant; the average level of FOXP3 in patients of similar age,
gender, race, graft-donor source, Banff histologic grade, or
initial antirejection treatment as the patient; a value for the
level of FOXP3 accepted in the art.
[0058] In an embodiment of the invention, generally, the level of
FOXP3 when compared to a control level may be increased by at least
about 10%, at least about 50%, or at least about 100%. The level of
FOXP3 when compared to a control level may be decreased by at least
about 10%, at least about 50%, or at least about 100% lower than
the level of FOXP3 in a control sample.
[0059] A control sample is typically the level of FOXP3 from a
healthy person or a person with a well-functioning (e.g., stable)
transplanted organ. A well-functioning (e.g., stable) transplanted
organ may be defined as a transplanted organ without acute
rejection, and preferable a transplanted organ that has not
developed transplant dysfunction or morphologic evidence of
transplant injury in areas of the transplant. For example, a stable
functioning kidney transplant may be defined as having a serum
creatinine concentration that has not changed by more than
approximately 0.2 mg per deciliter during the seven days before and
the seven days after collection of the biologic specimen for FOXP3
measurements.
[0060] It is not necessary to determine the level of FOXP3 mRNA or
FOXP3 protein in a control sample every time the method is
conducted. For example, the FOXP3 levels in the cell sample from
the transplanted organ or in the cell sample from the peripheral
blood can be compared to that of one or more previously determined
control samples or to a level recognized by the physician or
clinician conducting the method of a consensus of medical and/or
clinical practitioners.
EXAMPLES
Example 1
Methods
[0061] Study Cohoris. Urine samples from 83 kidney-transplant
recipients were examined. In this group were 36 subjects with graft
dysfunction (mean [.+-.SD] creatinine level, 3.6.+-.2.4 mg per
deciliter [318.2.+-.212.2 pmol per liter]) and biopsy-confirmed
acute rejection (mean age, 41.+-.12 years; 15 men and 21 women; 13
white, 12 black, and 11 with other racial or ethnic backgrounds;
with 20 living and 16 deceased donors), 29 subjects with stable
allograft function (mean creatinine level, 1.4.+-.0.4 mg per
deciliter [123.8.+-.35.4 .mu.mol per liter]) and normal allograft
biopsy (mean age, 44.+-.14 years; 15 men and 14 women; 12 white, 4
black, and 13 with other racial or ethnic backgrounds; with 26
living and 3 deceased donors), and 18 subjects with allograft
dysfunction (mean creatinine level, 3.1.+-.1.6 mg per deciliter
[274.0.+-.141.4 .mu.mol per liter]) and biopsies classified as
indicating chronic allograft nephropathy (mean age, 52.+-.12 years;
9 men and 9 women; 9 white, 2 black, and 7 with other racial or
ethnic backgrounds; with 5 living and 13 deceased donors).
[0062] Seventy-five of the 83 urine specimens were collected before
the biopsy procedure, and 8 samples were obtained after the
procedure. Formalin-fixed, paraffin-embedded renal-biopsy specimens
were stained with hematoxylin and eosin, periodic acid-Schiff, and
Masson's trichrome stains and were scored with the use of the Banff
97 classification by a pathologist who was blinded to the results
of molecular studies. Immunosuppression consisted of a calcineurin
inhibitor-based regimen (cyclosporine or tacrolimus), with the
administration of glucocorticoids, antilymphocyte antibodies
(muro-monab-CD3 [OKT3] or antithymocyte globulin), or both for the
treatment of acute rejection.
[0063] Quantitation of mRNA by Kinetic, Quantitative PCR. Total RNA
was isolated from urine-cell pellets, quantified and reverse
transcribed to complementary DNA (cDNA). Oligonucleotide primers
and fluorogenic probes were designed and synthesized for the
measurement of mRNA levels of FOXP3, CD25, CD3e, perforin, and 18S
ribosomal RNA (rRNA).
[0064] PCR analysis was performed by a two-step process, a
preamplification step followed by measurement of mRNA with an ABI
Prism 7700 system. Transcript levels were calculated by a standard
curve method, and mRNA copy numbers were normalized with the use of
18S rRNA copy numbers (the number of mRNA copies in 1 pg of RNA
divided by the number of 18S rRNA copies in 1 fg of RNA). When no
detectable level of a transcript was found, a value equal to half
the minimum observed 18S-normalized level was assigned. For an
estimation of group means, this method is considered a reasonable
substitute for the value of zero or the minimum detected value;
moreover, the nonparametric statistical tests of group differences
reported below are not affected by the choice of value.
[0065] Statistical Analysis. The levels of mRNA for FOXP3, CD25,
CD3.epsilon., perforin, and 18S rRNA deviated from a normal
distribution (P<0.001), but a log transformation substantially
reduced the positive skew. We used the 18S-normalized level as the
dependent variable in a Kruskal-Wallis test to identify any
differences among the group with acute rejection, the group with
chronic allograft nephropathy, and the group with normal biopsy
results and then used Dunn's test for multiple comparisons. The
Mann-Whitney test, equivalent to the Kruskal-Wallis test when
applied to two groups, was used when mRNA levels were compared
between two groups. Spearman's rank-order correlations were used to
test for a monotonic association of the 18S-adjusted mRNA
transcript levels with serum creatinine levels and time (in days)
from kidney transplantation to biopsy. An episode of acute
rejection was classified as reversible if the serum creatinine
level returned to within 15 percent of the prerejection level
within four weeks after the initiation of antirejection treatment.
A second end point was the loss of the graft during the first six
months after the diagnosis of acute rejection. We used
receiver-operating-characteristic (ROC) curves to analyze mRNA
levels in order to determine the cutoff points that yielded the
highest combined sensitivity and specificity for predicting the
outcome of an episode of acute rejection.
Example 2
Levels of FOXP3 mRNA in Urinary Cells
[0066] The log-transformed mean (.+-.SE) ratio of FOXP3 mRNA copies
to 18S-rRNA copies in urinary cells was 3.8.+-.0.5 in the 36
subjects with acute rejection and was higher than the levels in
both the 18 subjects with chronic allograft nephropathy
(1.3.+-.0.7) and the 29 subjects with normal biopsy results
(1.6.+-.0.4, P<0.001 by the Kruskal-Wallis test) (FIG. 1A).
Among the three groups, the 18S-normalized, log-transformed mRNA
levels of CD25 (6.9.+-.0.4, 4.0.+-.0.5, and 2.8.+-.0.6,
respectively; P<0.001), CD3.epsilon. (8.2.+-.0.4, 4.3.+-.0.5,
and 1.6.+-.0.5; P<0.001), and perforin (7.6.+-.0.4, 4.5.+-.0.4,
and 2.8.+-.0.4; P<0.001) were also highest in the
acute-rejection cohort (FIGS. 1B, 1C, and 1D).
Example 3
FOXP3 mRNA Levels and Disease Severity
[0067] We observed a significant inverse relationship between the
levels of FOXP3 mRNA and serum creatinine measured during an
episode of acute rejection (Spearman's correlation coefficient
[r.sub.s]=-0.38, P=0.02). By contrast, serum creatinine levels were
not significantly related to mRNA levels of CD25 (r.sub.s=-0.01,
P=0.93), CD3.epsilon. (r.sub.s=-0.11, P=0.54), or perforin
(r.sub.s=-0.23, P=0.18) in the acute-rejection group. Also, the
mean (.+-.SE) serum creatinine level in the 16 subjects with acute
rejection of Banff grade IA (moderate tubulitis) did not differ
significantly from that of the 20 subjects with grade IB (severe
tubulitis) or more (3.3.+-.0.6 mg per deciliter [291.7.+-.53.0
.mu.mol per liter] as compared with 3.8.+-.0.6 mg per deciliter
[318.2.+-.53.0 .mu.mol per liter], P=0.57).
[0068] There was no correlation between the levels of FOXP3 mRNA
and serum creatinine that were measured in the group with chronic
allograft nephropathy (r.sub.s=0.02, P=0.93) or the group with
normal biopsy results (r.sub.s=-0.08, P=0.67).
Example 4
FOXP3 mRNA Levels and Reversal of Acute Rejection
[0069] Twenty-six of the 36 episodes of acute rejection qualified
as successfully reversed; the remaining 10 did not. Levels of FOXP3
mRNA in urinary cells were significantly higher in the group with
successful reversal than in the group without reversal (mean
[.+-.SE] level, 4.7.+-.0.5 and 1.5.+-.0.7, respectively; P=0.001)
(FIG. 2A). In the two groups, the levels of mRNA for CD25
(7.3.+-.0.4 and 6.0.+-.0.9, P=0.22), CD3.epsilon. (8.5.+-.0.5 and
7.4.+-.0.8, P=0.35), and perforin (7.8.+-.0.5 and 7.3.+-.0.7,
P=0.43) were not informative of outcome (FIGS. 2B, 2C, and 2D).
[0070] The ROC curves (FIG. 3) show the fraction of true positive
results (sensitivity) and false positive results (1-specificity)
for various cutoff levels of mRNA for FOXP3, CD25, CD3.epsilon.,
and perforin. The log-transformed threshold that gave the maximal
sensitivity and specificity for FOXP3 mRNA was 3.46; using the
cutoff value of 3.46 derived from the data, the FOXP3 mRNA level
predicted rejection reversal with a sensitivity of 90 percent and a
specificity of 73 percent (P=0.001) (FIG. 3A). The levels of mRNA
for CD25, CD3.epsilon., and perform were not predictive of reversal
of acute rejection (FIGS. 3B, 3C, and 3D).
[0071] Successful reversal of acute rejection, as compared with
unsuccessful reversal, was not predicted by the subjects' age (mean
[.+-.SD], 41.+-.2.2 years and 40.+-.4.6 years, respectively;
P=0.68), sex (10 men and 16 women vs. 5 men and 5 women, P=0.71),
race (10 white, 6 black, and 10 with other race or ethnic
background vs. 3 white, 6 black, and 1 with other race or ethnic
background; P=0.08), graft-donor source (15 living and 11 deceased
vs. 5 living and 5 deceased, P=0.68), Banff histologic grade (11
with IA and 15 with >IA vs. 5 with IA and 5 with >IA,
P=0.68), or initial antirejection treatment (24 with
glucocorticoids and 2 with antilymphocyte antibodies vs. 7 with
glucocorticoids and 3 with antilymphocyte antibodies, P=0.12).
Among subjects with successful reversal, as compared with those
with unsuccessful reversal, serum creatinine levels (median levels,
2.3 mg per deciliter and 6.5 mg per deciliter, respectively;
P<0.001) and the time from kidney transplantation to the
development of acute rejection (median time, 82 days and 523 days,
respectively; P=0.008) were lower. In logistic-regression analyses
predicting nonresponse, levels of FOXP3 mRNA in urinary cells
remained significant after statistical control for serum creatinine
level (P=0.04) and the time from transplantation to rejection
(P=0.02).
[0072] A linear combination of levels of FOXP3 mRNA and creatinine
was a better predictor of rejection reversal (90 percent
sensitivity and 96 percent specificity) than FOXP3 mRNA levels
alone (90 percent sensitivity and 73 percent specificity) or serum
creatinine levels alone (85 percent sensitivity and 90 percent
specificity).
Example 5
FOXP3 mRNA Levels and Allograft Failure
[0073] Ten of the 36 subjects with acute rejection lost their
grafts within six months after the incident episode of acute
rejection, and 9 of those 10 subjects did not respond to the
initial antirejection therapy. Renal-allograft recipients with a
failed allograft within six months after the episode of acute
rejection had significantly lower FOXP3 mRNA levels in their
urinary cells than the 26 subjects who had a functioning allograft
(2.0 .+-.0.8 and 4.5.+-.0.5, respectively; P=0.001). In the two
groups, the levels of mRNA for CD25 (6.6.+-.0.7 and 7.1.+-.0.5,
P=0.33), CD3.epsilon. (7.9.+-.0.7 and 8.3.+-.0.5, P=0.76), and
perform (7.8.+-.0.6 and 7.6.+-.0.5, P=0.90) did not predict
allograft loss.
[0074] The rate and relative risk of graft failure within six
months after an episode of acute rejection, for thirds of each mRNA
measure, are shown in FIG. 4. At the highest third of FOXP3 mRNA
levels, the graft failure rate was 8 percent; at the middle third,
the graft failure rate was 25 percent and the relative risk was 3;
and at the lowest third, the graft failure rate was 50 percent and
the relative risk was 6 (P=0.02 by the chi-square test for linear
trend) (FIG. 4A). In contrast, the rate of graft failure after an
episode of acute rejection did not differ significantly across the
thirds of mRNA levels for CD25, CD3.epsilon., and perforin (FIGS.
4B, 4C, and 4D).
[0075] Graft failure as compared with graft success was not
predicted by the subjects' age (mean [.+-.SD] ages, 39.+-.4.2 years
and 42.+-.2.4 years, respectively; P=0.52), sex (4 men and 6 women
vs. 11 men and 15 women, P=0.90), race (3 white, 6 black, and 1
with other race or ethnic background vs. 10 white, 6 black, and 10
with other race or ethnic background; P=0.08), graft-donor source
(5 living and 5 deceased vs. 15 living and 11 deceased, P=0.68),
Banff histologic grade (5 with IA and 5 with >IA vs. 11 with IA
and 15 with >IA, P=0.68), or initial antirejection treatment (24
with glucocorticoids and 2 with antilymphocyte antibodies vs. 7
with glucocorticoids and 3 with antilymphocyte antibodies, P=0.12).
In subjects with graft failure, as compared with subjects with
graft success, serum creatinine levels (median levels, 6.5 mg per
deciliter [574.6 .mu.mol per liter] and 2.3 mg per deciliter [203.3
.mu.mol per liter], respectively; P<0.001) and the time from
kidney transplantation to the development of acute rejection
(median time, 562 days and 82 days; P=0.003) were significantly
greater. In a logistic-regression analysis, FOXP3 mRNA levels
became nonsignificant after control for serum creatinine levels
(P=0.13) or time between transplantation and rejection
(P=0.09).
[0076] A linear combination of levels of FOXP3 mRNA and creatinine
was a better predictor of graft failure (90 percent sensitivity and
92 percent specificity) than were either FOXP3 mRNA levels alone
(80 percent sensitivity and 69 percent specificity) or serum
creatinine levels alone (85 percent sensitivity and 90 percent
specificity).
Example 6
FOXP3 mRNA Levels and Time to Acute Rejection
[0077] Late acute rejection (acute rejection occurring at least
three months after transplantation) results in an outcome that is
inferior to that of early acute rejection. We found a strong
inverse relationship between levels of FOXP3 mRNA in urinary cells
and the time from kidney transplantation to the development of
acute rejection (r.sub.s=-0.42, P=0.01) (FIG. 5A). Levels of FOXP3
mRNA in urinary cells were lower in 11 urine specimens from
patients with late acute rejection than in 25 specimens from
patients with early acute rejection (mean [.+-.SE] level,
2.5.+-.0.6 and 4.7.+-.0.5; P=0.009). CD25 mRNA levels also showed
an inverse relation (r.sub.s=-0.45, P=0.006) (FIG. 5B), and the
levels were lower during late rejection than during early acute
rejection (5.8.+-.0.8 and 7.4.+-.0.4, P=0.07). There was no inverse
relation between the time from kidney transplantation to the
development of acute rejection and the mRNA levels of CD3.epsilon.
(r.sub.s=-0.26, P=0.12) (FIG. 5C) or perforin (r.sub.s=-0.02,
P=0.91) (FIG. 5D). There was also no correlation between the serum
creatinine levels at the time of acute rejection and the time from
kidney transplantation to the development of acute rejection
(r.sub.s=0.23, P=0.17).
Example 7
FOXP3 Levels in Cell Samples from Peripheral Blood are Predictive
of Acute Rejection Outcome
[0078] To determine whether peripheral blood cell FOXP3 mRNA levels
are informative of human renal allograft status, peripheral blood,
using PAXgene RNA tubes, were collected from 38 renal allograft
recipients; 11 subjects with acute rejection and 27 subjects with
normal protocol biopsies and stable graft function. Peripheral
blood cell mRNA for FOXP3 and mRNA for a constitutively expressed
gene 18S ribosomal RNA (18S rRNA) were measured using kinetic
quantitative PCR assay. The level of expression, normalized using
18S rRNA copy numbers and log transformed to reduce the positive
skew, was correlated with renal allograft status.
[0079] Data analyses demonstrated that peripheral blood cell FOXP3
mRNA levels are significantly lower during an episode of acute
rejection as compared to levels observed in samples from subjects
with normal biopsies and stable graft function (P=0.04, Mann
Whitney test). Furthermore, graft outcome following an episode of
acute rejection was predicted by peripheral blood cell FOXP3 mRNA
levels; there was no graft loss in the lowest tertile, 3 of 4
grafts were lost in the middle tertile, and 2 of 3 grafts failed in
the highest tertile.
* * * * *