U.S. patent number RE47,057 [Application Number 13/529,768] was granted by the patent office on 2018-09-25 for methods and compositions for evaluating graft survival in a solid organ transplant recipient.
This patent grant is currently assigned to The Board of Trustees of the Leland Stanford Junior University. The grantee listed for this patent is Elaine S. Mansfield, Minnie M. Sarwal. Invention is credited to Elaine S. Mansfield, Minnie M. Sarwal.
United States Patent |
RE47,057 |
Sarwal , et al. |
September 25, 2018 |
**Please see images for:
( Certificate of Correction ) ** |
Methods and compositions for evaluating graft survival in a solid
organ transplant recipient
Abstract
Methods are provided for evaluating a subject for graft
survival, e.g., in terms of predicting graft survival, identifying
the presence of a deleterious graft condition, such as CAN and DT,
identifying the severity and class of acute rejection, etc, in a
subject are provided. In practicing the subject methods, the
expression of at least one gene in a sample from the subject, e.g.,
a blood or biopsy sample, is assayed, e.g., at the nucleic acid
and/or protein level, to evaluate the subject. Also provided are
compositions, systems and kits that find use in practicing the
subject methods. The methods and compositions find use in a variety
of applications.
Inventors: |
Sarwal; Minnie M. (Portola
Valley, CA), Mansfield; Elaine S. (Sunnyvale, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Sarwal; Minnie M.
Mansfield; Elaine S. |
Portola Valley
Sunnyvale |
CA
CA |
US
US |
|
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University (Stanford, CA)
|
Family
ID: |
36992379 |
Appl.
No.: |
13/529,768 |
Filed: |
June 21, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
60662083 |
Mar 14, 2005 |
|
|
|
Reissue of: |
11375681 |
Mar 13, 2006 |
7741038 |
Jun 22, 2010 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6876 (20130101); C12Q 1/6883 (20130101); C12Q
1/6883 (20130101); C12Q 1/6876 (20130101); C12Q
2600/158 (20130101); C12Q 2600/106 (20130101); C12Q
2600/118 (20130101); C12Q 2600/158 (20130101); C12Q
2600/106 (20130101); C12Q 2600/118 (20130101) |
Current International
Class: |
C12Q
1/68 (20180101); G01N 33/53 (20060101); C12Q
1/6883 (20180101); C12Q 1/6876 (20180101) |
References Cited
[Referenced By]
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EP |
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2007/104537 |
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|
Primary Examiner: Railey; Johnny F
Attorney, Agent or Firm: Gurley; Kyle A. Field; Bret E.
Bozicevic, Field & Francis LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
.Iadd.Notice: More than one reissue application has been filed for
the reissue of U.S. Pat. No. 7,741,038. The reissue applications
are application Ser. No. 13/529,768 (the present application), and
Ser. No. 13/943,626, filed Jul. 16, 2013 (a continuation reissue
application). .Iaddend.
.[.Pursuant to 35 U.S.C. .sctn.119 (e), this application.].
.Iadd.This application is a Reissue of U.S. Pat. No. 7,741,038,
which patent issued on Jun. 22, 2010, and which patent
.Iaddend.claims priority to the filing date of U.S. Provisional
Patent Application Ser. No. 60/662,083 filed on Mar. 14, 2005; the
disclosure of which application is herein incorporated by
reference.
Claims
What is claimed is:
1. A method of evaluating .[.graft survival in a subject.].
.Iadd.gene expression levels of at least two genes in a sample from
a transplant recipient.Iaddend., said method comprising:
.Iadd.providing a sample from a transplant recipient; and .Iaddend.
.[.assessing.]. .Iadd.measuring an amount of .Iaddend.expression of
at least two genes in .[.a.]. .Iadd.the .Iaddend.sample .[.from
said subject to evaluate graft survival in said subject.]., wherein
said at least two genes comprises .[.HIST1H2B.]. .Iadd.HIST1H2BC
.Iaddend.and IGHG3.
2. The method according to claim 1, wherein said expression of at
least two genes is assessed by assaying said sample for a nucleic
acid transcript of said gene.
3. The method according to claim 1, wherein said expression of at
least two genes is assessed by assaying said sample for an
expression product of said gene.
4. The method according to .[.any of.]. claim 1, wherein said
sample is a blood sample.
5. The method according to claim 4, wherein said blood sample is a
peripheral blood sample.
6. The method according to claim 1, wherein said sample is a tissue
biopsy sample.
7. A method according to claim 1, wherein the method comprises:
obtaining an expression profile for a sample from said subject.
.[.8. The method according to claim 7, wherein said expression
profile is compared to a reference expression profile..].
9. The method according to claim .[.8.]. .Iadd.7.Iaddend., wherein
said expression profile is a nucleic acid expression profile.
10. The method according to claim .[.8.]. .Iadd.7.Iaddend., wherein
said expression profile comprises expression measurements for at
least 5 different genes.
11. The method according to claim .[.8.]. .Iadd.7.Iaddend., wherein
said expression profile is determined using a microarray.
12. The method according to claim 11, wherein said microarray is a
genomic array.
13. A method of .[.managing post-transplantation therapy in a
subject, said method.]. .Iadd.treating a transplant recipient
.Iaddend.comprising: (a) .[.evaluating.]. .Iadd.determining that a
transplant recipient has a .Iaddend.graft survival .[.in said
subject by a method according to claim 1; and.]. .Iadd.phenotype by
evaluating results previously obtained from a quantitative
determination of nucleic acid expression levels of at least two
genes in a sample from the transplant recipient, and treating said
transplant recipient by maintaining a current therapeutic regimen;
or .Iaddend. (b) determining .[.a post-transplantation therapy
protocol based on said evaluation step (a);.]. .Iadd.that a
transplant recipient has a graft loss phenotype by evaluating
results previously obtained from a quantitative determination of
nucleic acid expression levels of at least two genes in a sample
from the transplant recipient, and treating said transplant
recipient by increasing or decreasing a therapeutic regimen;
wherein, said evaluating comprises comparing said results to a
reference nucleic acid expression profile comprising said at least
two genes; and .Iaddend. .[.to manage post-transplantation therapy
in said subject.]. .Iadd.wherein said at least two genes comprises
HIST1H2BC and IGHG3.Iaddend..
14. The method according to claim 13, wherein said subject is a
human.
15. The method according to claim 1, wherein said at least two
genes further comprises one or more genes selected from: AHSA2,
TNFRSF10D, MAPK9, IFNAR2, TM4SF9, MIF, SCYE1, MAPK1, TGFBR3, IGKC,
IL1R2 and IGL.
.Iadd.16. The method of claim 7, wherein said expression profile
comprises expression measurements for at least ten different genes.
.Iaddend.
.Iadd.17. A method of assaying gene expression in a blood sample
from a graft recipient, the method comprising: a) receiving a
sample of blood from a patient that has received a graft; and b)
assaying the expression of at least two genes in the blood sample,
wherein said at least two genes comprises HIST1H2BC and IGHG3.
.Iaddend.
.Iadd.18. The method according to claim 13, wherein the therapeutic
regimen is an immunosuppressive therapy. .Iaddend.
.Iadd.19. The method according to claim 13, comprising: determining
that the transplant recipient has a graft loss phenotype that is
calcineurin-inhibitor drug nephrotoxicity (DT); and decreasing an
immunosuppressive therapy. .Iaddend.
.Iadd.20. The method according to claim 13, comprising: (i)
determining that the transplant recipient has a graft loss
phenotype that is chronic allograft nephropathy (CAN); and (ii)
increasing an immunosuppressive therapy, or changing an
immunosuppressive therapy by administering a different
immunosuppressive drug. .Iaddend.
.Iadd.21. The method according to claim 1, further comprising
measuring an amount of expression of control genes in the sample.
.Iaddend.
Description
BACKGROUND
Transplantation of a graft organ or tissue from a donor to a host
patient is a feature of certain medical procedures and treatment
protocols. Despite efforts to avoid graft rejection through
host-donor tissue type matching, in transplantation procedures
where a donor organ is introduced into a host, immunosuppressive
therapy is generally required to the maintain viability of the
donor organ in the host.
After an organ has been transplanted into the patient, the
patient's immune system is suppressed to prevent rejection of the
new organ. Despite the wide use of immunosuppressive therapy, organ
transplant rejection can occur.
Organ transplant rejection comprises three separate categories:
hyperacute, acute and chronic. Hyperacute rejection is
characterized by rapid thrombotic occlusion of the graft
vasculature within minutes to hours after organ transplantation.
Hyperacute rejection is mediated in large part by preexisting
antibodies that bind to the epithelium and activate the complement
cascade. Complement activation results in endothelial cell damage
and subsequent exposure of the basement membrane, resulting in the
activation of platelets, leading to thrombosis and vascular
occlusion. As the field of transplantation has matured, hyperacute
rejection has become less common due to blood antigen and MHC
molecule matching between the donor organ and the recipient.
Acute rejection is sub-classified into acute vascular rejection and
acute cellular rejection. Acute vascular rejection is characterized
by necrosis of individual cells in the graft blood vessels. The
process is similar to that of hyperacute rejection, but onset is
often slower, within one week of rejection, and a T cell component
may be involved. Acute vascular rejection is initiated by a
response to alloantigens present on the vascular endothelial cells
of the donor organ, resulting in the release of a cytokine cascade,
inflammation, and eventual necrosis. Acute cellular rejection is
often characterized by necrosis of the essential or parenchymal
cells of the transplanted organ caused by the infiltration of host
T lymphocytes and macrophages. The lymphocytes involved are usually
cytotoxic T lymphocytes (CTL) and macrophages, both resulting in
lysis of targeted cells. The CTLs are usually specific for graft
alloantigens displayed in the context of MHC class I molecules.
Chronic rejection is the major cause of allograft loss and is
characterized by fibrosis and loss of normal organ structures.
Fibrosis may be the result of wound healing following the cellular
necrosis of acute rejection, or may occur independently and without
prior acute rejection. In addition, chronic rejection may lead to
vascular occlusions thought to stem from a delayed type
hypersensitivity response to alloantigens present on the
transplanted organ. These alloantigens stimulate lymphocytes to
secrete cytokines which attract macrophages and other effector
cells eventually leading to an arteriosclerosis-like blockage.
In many cases, chronic graft injury or rejection (CR) is largely
due to calcineurin-inhibitor drug nephrotoxicity (DT) and chronic
allograft nephropathy (CAN), two conditions which may result in
loss of graft function and early graft loss, premature to the life
expectancy of the recipient. The incidence of chronic graft loss
has remained unchanged over the last decade.
A biopsy is the only current gold standard for CAN and DT
diagnosis. As both conditions are progressive post-transplantation,
multiple graft protocol biopsies are required. However, the
invasiveness of biopsy procedures is a limitation to this form of
monitoring. In addition, variability of biopsy sampling and
pathology analysis (2) adds a confounder to the differential
diagnosis of these 2 conditions--the result of either too much drug
(DT) vs. too little/inappropriate drugs (CAN)--with a common
outcome of chronic fibrotic injury from differing mechanisms
(non-immune vs. immune).
There is currently no method available to detect or to monitor
future graft loss at the time of transplantation or acute rejection
(AR) episodes. AR is a risk factor both for eventual graft loss,
delayed recovery of graft function and even chronic rejection.
Non-invasive monitoring methods for AR stratification, CR, DT and
developing or established tolerance is currently not available, but
would be very valuable, as the transplant biopsy, though the
current gold standard, fails to stratify or prognosticate AR,
differentiate CR clearly from DT or diagnose tolerance.
Accordingly, of interest would be the ability to evaluate
likelihood of graft survival in a transplant recipient, e.g.,
following an AR episode, such that treatment protocols for
transplant patients may be customized.
SUMMARY OF THE INVENTION
Methods are provided for evaluating a subject for graft survival,
e.g., in terms of predicting graft survival, identifying the
presence of a deleterious graft condition, such as CAN and DT,
identifying the severity and class of acute rejection, etc, in a
subject are provided. In practicing the subject methods, the
expression of at least one gene in a sample from the subject, e.g.,
a blood or biopsy sample, is assayed, e.g., at the nucleic acid
and/or protein level, to evaluate the subject. Also provided are
compositions, systems and kits that find use in practicing the
subject methods.
DEFINITIONS
For convenience, certain terms employed in the specification,
examples, and appended claims are collected here.
"Acute rejection or AR" is the rejection by the immune system of a
tissue transplant recipient when the transplanted tissue is
immunologically foreign. Acute rejection is characterized by
infiltration of the transplanted tissue by immune cells of the
recipient, which carry out their effector function and destroy the
transplanted tissue. The onset of acute rejection is rapid and
generally occurs in humans within a few weeks after transplant
surgery. Generally, acute rejection can be inhibited or suppressed
with immunosuppressive drugs such as rapamycin, cyclosporin A,
anti-CD40L monoclonal antibody and the like.
"Chronic transplant rejection or CR" generally occurs in humans
within several months to years after engraftment, even in the
presence of successful immunosuppression of acute rejection.
Fibrosis is a common factor in chronic rejection of all types of
organ transplants. Chronic rejection can typically be described by
a range of specific disorders that are characteristic of the
particular organ. For example, in lung transplants, such disorders
include fibroproliferative destruction of the airway (bronchiolitis
obliterans); in heart transplants or transplants of cardiac tissue,
such as valve replacements, such disorders include fibrotic
atherosclerosis; in kidney transplants, such disorders include,
obstructive nephropathy, nephrosclerorsis, tubulointerstitial
nephropathy; and in liver transplants, such disorders include
disappearing bile duct syndrome. Chronic rejection can also be
characterized by ischemic insult, denervation of the transplanted
tissue, hyperlipidemia and hypertension associated with
immunosuppressive drugs.
The term "transplant rejection" encompasses both acute and chronic
transplant rejection.
The term "stringent assay conditions" as used herein refers to
conditions that are compatible to produce binding pairs of nucleic
acids, e.g., surface bound and solution phase nucleic acids, of
sufficient complementarity to provide for the desired level of
specificity in the assay while being less compatible to the
formation of binding pairs between binding members of insufficient
complementarity to provide for the desired specificity. Stringent
assay conditions are the summation or combination (totality) of
both hybridization and wash conditions.
"Stringent hybridization conditions" and "stringent hybridization
wash conditions" in the context of nucleic acid hybridization
(e.g., as in array, Southern or Northern hybridizations) are
sequence dependent, and are different under different experimental
parameters. Stringent hybridization conditions that can be used to
identify nucleic acids within the scope of the invention can
include, e.g., hybridization in a buffer comprising 50% formamide,
5.times.SSC, and 1% SDS at 42.degree. C., or hybridization in a
buffer comprising 5.times.SSC and 1% SDS at 65.degree. C., both
with a wash of 0.2.times.SSC and 0.1% SDS at 65.degree. C.
Exemplary stringent hybridization conditions can also include
hybridization in a buffer of 40% formamide, 1 M NaCl, and 1% SDS at
37.degree. C., and a wash in 1.times.SSC at 45.degree. C.
Alternatively, hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. can be employed. Yet additional stringent hybridization
conditions include hybridization at 60.degree. C. or higher and
3.times.SSC (450 mM sodium chloride/45 mM sodium citrate) or
incubation at 42.degree. C. in a solution containing 30% formamide,
1M NaCl, 0.5% sodium sarcosine, 50 mM MES, pH 6.5. Those of
ordinary skill will readily recognize that alternative but
comparable hybridization and wash conditions can be utilized to
provide conditions of similar stringency.
In certain embodiments, the stringency of the wash conditions that
set forth the conditions which determine whether a nucleic acid is
specifically hybridized to a surface bound nucleic acid. Wash
conditions used to identify nucleic acids may include, e.g.: a salt
concentration of about 0.02 molar at pH 7 and a temperature of at
least about 50.degree. C. or about 55.degree. C. to about
60.degree. C.; or, a salt concentration of about 0.15 M NaCl at
72.degree. C. for about 15 minutes; or, a salt concentration of
about 0.2.times.SSC at a temperature of at least about 50.degree.
C. or about 55.degree. C. to about 60.degree. C. for about 15 to
about 20 minutes; or, the hybridization complex is washed twice
with a solution with a salt concentration of about 2.times.SSC
containing 0.1% SDS at room temperature for 15 minutes and then
washed twice by 0.1.times.SSC containing 0.1% SDS at 68.degree. C.
for 15 minutes; or, equivalent conditions. Stringent conditions for
washing can also be, e.g., 0.2.times.SSC/0.1% SDS at 42.degree.
C.
A specific example of stringent assay conditions is rotating
hybridization at 65.degree. C. in a salt based hybridization buffer
with a total monovalent cation concentration of 1.5 M (e.g., as
described in U.S. patent application Ser. No. 09/655,482 filed on
Sep. 5, 2000, the disclosure of which is herein incorporated by
reference) followed by washes of 0.5.times.SSC and 0.1.times.SSC at
room temperature.
Stringent assay conditions are hybridization conditions that are at
least as stringent as the above representative conditions, where a
given set of conditions are considered to be at least as stringent
if substantially no additional binding complexes that lack
sufficient complementarity to provide for the desired specificity
are produced in the given set of conditions as compared to the
above specific conditions, where by "substantially no more" is
meant less than about 5-fold more, typically less than about 3-fold
more. Other stringent hybridization conditions are known in the art
and may also be employed, as appropriate.
As used herein, the term "gene" or "recombinant gene" refers to a
nucleic acid comprising an open reading frame encoding a
polypeptide, including exon and (optionally) intron sequences. The
term "intron" refers to a DNA sequence present in a given gene that
is not translated into protein and is generally found between exons
in a DNA molecule. In addition, a gene may optionally include its
natural promoter (i.e., the promoter with which the exons and
introns of the gene are operably linked in a non-recombinant cell,
i.e., a naturally occurring cell), and associated regulatory
sequences, and may or may not have sequences upstream of the AUG
start site, and may or may not include untranslated leader
sequences, signal sequences, downstream untranslated sequences,
transcriptional start and stop sequences, polyadenylation signals,
translational start and stop sequences, ribosome binding sites, and
the like.
A "protein coding sequence" or a sequence that "encodes" a
particular polypeptide or peptide, is a nucleic acid sequence that
is transcribed (in the case of DNA) and is translated (in the case
of mRNA) into a polypeptide in vitro or in vivo when placed under
the control of appropriate regulatory sequences. The boundaries of
the coding sequence are determined by a start codon at the 5'
(amino) terminus and a translation stop codon at the 3' (carboxy)
terminus. A coding sequence can include, but is not limited to,
cDNA from viral, procaryotic or eukaryotic mRNA, genomic DNA
sequences from viral, procaryotic or eukaryotic DNA, and even
synthetic DNA sequences. A transcription termination sequence may
be located 3' to the coding sequence.
The terms "reference" and "control" are used interchangebly to
refer to a known value or set of known values against which an
observed value may be compared. As used herein, known means that
the value represents an understood parameter, e.g., a level of
expression of a marker gene in a graft survival or loss
phenotype.
The term "nucleic acid" includes DNA, RNA (double-stranded or
single stranded), analogs (e.g., PNA or LNA molecules) and
derivatives thereof. The terms "ribonucleic acid" and "RNA" as used
herein mean a polymer composed of ribonucleotides. The terms
"deoxyribonucleic acid" and "DNA" as used herein mean a polymer
composed of deoxyribonucleotides. The term "mRNA" means messenger
RNA. An "oligonucleotide" generally refers to a nucleotide multimer
of about 10 to 100 nucleotides in length, while a "polynucleotide"
includes a nucleotide multimer having any number of
nucleotides.
The terms "protein" and "polypeptide" used in this application are
interchangeable. "Polypeptide" refers to a polymer of amino acids
(amino acid sequence) and does not refer to a specific length of
the molecule. Thus peptides and oligopeptides are included within
the definition of polypeptide. This term does also refer to or
include post-translational modifications of the polypeptide, for
example, glycosylations, acetylations, phosphorylation and the
like. Included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid, polypeptides with
substituted linkages, as well as other modifications known in the
art, both naturally occurring and non-naturally occurring.
The term "assessing" and "evaluating" are used interchangeably to
refer to any form of measurement, and includes determining if an
element is present or not. The terms "determining," "measuring,"
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations. Assessing may be
relative or absolute. "Assessing the presence of" includes
determining the amount of something present, as well as determining
whether it is present or absent.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Predictive Analysis of Microarrays (PAM) using a set of
3,170 differentially expressed genes identifies the 33 classifiers
with similar power (FIG. 1A). The PAM classification scores grouped
the samples with 100% concordance to assigned classes and reported
scores are aligned with the clustered samples (FIG. 1B).
FIG. 2. Kaplan-Meier survival analysis for graft loss (red) and
no-loss (blue). The genes include ICAM5 (FIG. 2A; p=0.007), IL6R
(FIG. 2B; p=0.003), STAT1 (FIG. 2C; p=0.036), and STAT6 (FIG. 2D
(p=0.020).
FIG. 3. Kaplan-Meier survival curves for 8 genes from whole blood
samples that are predictive of graft loss. Genes include AHSA2
(FIG. 3A), IGHG1 (FIG. 3B), IFNAR2 (FIG. 3C), IGKC (FIG. 3D),
HIST1H2BC (FIG. 3E), IL1R2 (FIG. 3F), MAPK1 (FIG. 3G), and MAPK9
(FIG. 3H).
FIG. 4. Demonstrates that gene expression is generally
uniform/consistent across the full clinical groups analyzed as the
gene expression levels segregate well within patient groups.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods are provided for evaluating a subject for graft function,
e.g., in terms of predicting graft survival, identifying the
presence of a deleterious graft condition, such as CAN and DT,
identifying the severity and class of acute rejection, etc, in a
subject are provided. In practicing the subject methods, the
expression of at least one gene in a sample from the subject, e.g.,
a blood or biopsy sample, is assayed, e.g., at the nucleic acid
and/or protein level, to evaluate the subject. Also provided are
compositions, systems and kits that find use in practicing the
subject methods. The methods and compositions find use in a variety
of applications.
Before the present invention is described in greater detail, it is
to be understood that this invention is not limited to particular
embodiments described, as such may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
Where a range of values is provided, it is understood that each
intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
All publications and patents cited in this specification are herein
incorporated by reference as if each individual publication or
patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
It is noted that, as used herein and in the appended claims, the
singular forms "a", "an", and "the" include plural referents unless
the context clearly dictates otherwise. It is further noted that
the claims may be drafted to exclude any optional element. As such,
this statement is intended to serve as antecedent basis for use of
such exclusive terminology as "solely," "only" and the like in
connection with the recitation of claim elements, or use of a
"negative" limitation.
As will be apparent to those of skill in the art upon reading this
disclosure, each of the individual embodiments described and
illustrated herein has discrete components and features which may
be readily separated from or combined with the features of any of
the other several embodiments without departing from the scope or
spirit of the present invention. Any recited method can be carried
out in the order of events recited or in any other order which is
logically possible.
As summarized above, the subject invention is directed to methods
of evaluating graft function in a subject, as well as reagents and
kits for use in practicing the subject methods. In further
describing the invention, the subject methods are described first,
followed by a review of the reagents and kits for use in practicing
the subject methods.
Methods of Evaluating Graft Function
As reviewed above, the subject invention provides methods for
evaluating a subject for graft survival. The methods provide for
evaluating a subject for graft survival in terms of a number of
different factors. In certain embodiments, the factor evaluated is
a basic prediction of graft survival. In certain embodiments, the
factor evaluated is the presence of a deleterious graft condition,
such as CAN and DT. In certain embodiments, the factor identified
is the severity and/or class of acute rejection, where these
embodiments are distinguished from methods that just identify the
presence of acute rejection, since one is further determining the
severity and/or class of acute rejection, and therefore an aspect
of graft survival
As such, certain embodiments of the invention provide methods of
evaluating, e.g., in terms of predicting, graft survival in a
subject comprising a graft. As such, the subject invention provides
methods of evaluating whether a graft in a transplant patient or
subject will survive or be lost. In certain embodiments, the
methods may be viewed as methods of determining whether a
transplant subject has a graft survival phenotype, i.e., a
phenotype in which the graft will survive. A graft survival
phenotype is a phenotype characterized by the presence of long-term
graft survival. By "long-term" graft survival is meant graft
survival for at least about 5 years beyond current sampling,
despite the occurrence of one or more prior episodes of AR. In
certain embodiments, graft survival is determined for patients in
which at least one episode of acute rejection (AR) has occurred. As
such, these embodiments are methods of determining or predicting
graft survival following AR. Graft survival is determined or
predicted in certain embodiments in the context of transplant
therapy, e.g., immunosuppressive therapy, where immunosuppressive
therapies are known in the art. In yet other embodiments, methods
of distinguishing being organ rejection disease conditions, such as
CAN and DT, are provided. In yet other embodiments, methods of
determining the class and/or severity of acute rejection (and not
just the presence thereof are provided.
As in known in the transplantation field, the graft organ, tissue
or cell(s) may be allogeneic or xenogeneic, such that the grafts
may be allografts or xenografts. Organs and tissues of interest
include, but are not limited to: skin, heart, kidney, liver, bone
marrow, and other organs.
In practicing the subject methods, a subject or patient sample,
e.g., cells or collections thereof, e.g., tissues, is assayed to
evaluate graft survival in the host, e.g., whether the graft will
survive in the host from which the assayed sample was obtained.
Accordingly, the first step of the subject methods is to obtain a
suitable sample from the subject or patient of interest, i.e., a
patient having at least one graft, e.g., allograft.
The sample is derived from any initial suitable source, where
sample sources of interest include, but are not limited to, many
different physiological sources, e.g., CSF, urine, saliva, tears,
tissue derived samples, e.g., homogenates (such as biopsy samples
of the transplanted tissue or organ (including, but not limited to
kidney, heart, lung biopsies), and blood or derivatives
thereof.
In certain embodiments, a suitable initial source for the patient
sample is blood. As such, the sample employed in the subject assays
of these embodiments is generally a blood-derived sample. The blood
derived sample may be derived from whole blood or a fraction
thereof, e.g., serum, plasma, etc., where in certain embodiments
the sample is derived from blood cells harvested from whole blood.
Of particular interest as a sample source are peripheral blood
lymphocytes (PBL). Any convenient protocol for obtaining such
samples may be employed, where suitable protocols are well known in
the art and a representative protocol is reported in the
Experimental Section, below.
In practicing the subject methods, the sample is assayed to obtain
an expression evaluation, e.g., expression profile, for one or more
genes, where the term expression profile is used broadly to include
a genomic expression profile, e.g., an expression profile of
nucleic acid transcripts, e.g., mRNAs, of the one or more genes of
interest, or a proteomic expression profile, e.g., an expression
profile of one or more different proteins, where the
proteins/polypeptides are expression products of the one or more
genes of interest. As such, in certain embodiments the expression
of only one gene is evaluated. In yet other embodiments, the
expression of two or more, e.g., about 5 or more, about 10 or more,
about 15 or more, about 25 or more, about 50 or more, about 100 or
more, about 200 or more, etc., genes is evaluated. Accordingly, in
the subject methods, the expression of at least one gene in a
sample is evaluated. In certain embodiments, the evaluation that is
made may be viewed as an evaluation of the transcriptosome, as that
term is employed in the art. See e.g., Gomes et al., Blood (2001
Jul. 1) 98(1): 93-9.
In generating the expression profile, in certain embodiments a
sample is assayed to generate an expression profile that includes
expression data for at least one gene/protein, usually a plurality
of genes/proteins, where by plurality is meant at least two
different genes/proteins, and often at least about 5, typically at
least about 10 and more usually at least about 20 different
genes/proteins or more, such as 50 or more, 100 or more, etc.
In the broadest sense, the expression evaluation may be qualitative
or quantitative. As such, where detection is qualitative, the
methods provide a reading or evaluation, e.g., assessment, of
whether or not the target analyte, e.g., nucleic acid or expression
product, is present in the sample being assayed. In yet other
embodiments, the methods provide a quantitative detection of
whether the target analyte is present in the sample being assayed,
i.e., an evaluation or assessment of the actual amount or relative
abundance of the target analyte, e.g., nucleic acid in the sample
being assayed. In such embodiments, the quantitative detection may
be absolute or, if the method is a method of detecting two or more
different analytes, e.g., target nucleic acids, in a sample,
relative. As such, the term "quantifying" when used in the context
of quantifying a target analyte, e.g., nucleic acid(s), in a sample
can refer to absolute or to relative quantification. Absolute
quantification may be accomplished by inclusion of known
concentration(s) of one or more control analytes and referencing
the detected level of the target analyte with the known control
analytes (e.g., through generation of a standard curve).
Alternatively, relative quantification can be accomplished by
comparison of detected levels or amounts between two or more
different target analytes to provide a relative quantification of
each of the two or more different analytes, e.g., relative to each
other.
Genes/proteins of interest are graft survival/loss indicative
genes, i.e., genes/proteins that are differentially expressed or
present at different levels in graft survival and graft loss
individuals (more specifically, individuals in which graft loss
will occur vs. individuals in which a graft will survive).
Representative genes/proteins of interest in certain embodiments
include, but are not limited to, the genes/proteins provided in
Tables 1 and 2. (Note that for Tables 1 and 2, the exact sequence
of the clone identified in the table can be determined through the
NCBI Entrez nucleotide database located at the website produced by
placing "http://www." before:
"ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&db=nucleotide"
in the navigation window of a web browser (e.g., Netscape); the
sequence for a specific clone is then obtained by entering the
clone ID in quotes as the search term).
TABLE-US-00001 TABLE 1 Genes of known function in whole blood
predictive of graft loss following acute rejection Rank Clone
Symbol Gene UnigeneID 1 IMAGE: 214006 HIST1H2BC Histone 1, H2bc
Hs.356901 2 IMAGE: 826131 IGHG3 Ig heavy constant gamma 3 Hs.413826
3 IMAGE: 626318 UBN1 Ubinuclein 1 Hs.21479 4 IMAGE: 511387 GLG1
Golgi apparatus protein 1 Hs.78979 5 IMAGE: 810057 CSDA Cold shock
domain protein A Hs.221889 6 IMAGE: 283919 HIST1H2AC Histone 1,
H2ac Hs.28777 7 IMAGE: 453710 PLEK2 Pleckstrin 2 Hs.170473 8 IMAGE:
840821 SSR4 Signal sequence receptor, delta Hs.409223 9 IMAGE:
70201 MSCP Mitochondrial solute carrier Hs.283716 10 IMAGE: 66686
RPL10 Ribosomal protein L10 Hs.77091 11 IMAGE: 1306420 AHSA2
Activator of heat shock ATPase Hs.122440 12 IMAGE: 2578221 UBB
Ubiquitin B Hs.356190 13 IMAGE: 811062 CGI-69 CGI-69 protein
Hs.237924 14 IMAGE: 1272566 TNFRSF10D TNF receptor superfamily 10d
Hs.129844 15 IMAGE: 1240649 RPL10 Ribosomal protein L10 Hs.77091 16
IMAGE: 85224 RBM25 RNA binding motif protein 25 Hs.197184 17 IMAGE:
2114004 HIST1H3D Histone 1, H3d Hs.239458 18 IMAGE: 789091
HIST1H2AC Histone 1, H2ac Hs.28777 19 IMAGE: 591025 JMJD3 Jumonji
domain containing 3 Hs.103915 20 IMAGE: 1354406 SSR4 Signal
sequence receptor, delta Hs.409223 21 IMAGE: 812276 SNCA Synuclein
Hs.76930 22 IMAGE: 344720 GYPC Glycophorin C Hs.81994 23 IMAGE:
683899 JMJD3 Jumonji domain containing 3 Hs.103915 24 IMAGE: 825006
CYorf15A Chromosome Y ORF Hs.171857 25 IMAGE: 1492412 UBA52
Ubiquitin A-52 fusion product 1 Hs.5308 26 IMAGE: 854079 ACTN1
Actinin, alpha 1 Hs.119000 27 IMAGE: 366884 IFNAR2 Interferon (a-
B- and o) receptor 2 Hs.86958 28 IMAGE: 812967 TM4SF9 Transmembrane
4 superfamily Hs.8037 29 IMAGE: 207794 NFE2 Erythroid nuclear
factor Hs.75643 30 IMAGE: 359835 SAT Spermidine
N1-acetyltransferase Hs.28491 31 IMAGE: 565849 KLHL12 Kelch-like 12
(Drosophila) Hs.3826 32 IMAGE: 256260 RFC3 Replication factor C
activator Hs.115474 33 IMAGE: 191826 MSCP Mitochondrial solute
carrier protein Hs.283716 34 IMAGE: 202242 MIF Macrophage migration
inhibitor Hs.407995 35 IMAGE: 323506 MAPK1 Mitogen-activated
protein kinase 1 Hs.324473 36 IMAGE: 1286850 MME Membrane
metallo-endopeptidase Hs.259047 37 IMAGE: 129725 RBPSUH Recombining
binding protein Hs.347340 38 IMAGE: 882522 ASS Argininosuccinate
synthetase Hs.160786 39 IMAGE: 2129439 UBE2B Ubiquitin-conjugating
enzyme E2B Hs.385986 40 IMAGE: 1687138 HIST1H2AM Histone 1, H2am
Hs.134999 41 IMAGE: 209655 TGFBR3 TGFb receptor III Hs.342874 42
IMAGE: 75254 CSRP2 Cysteine and glycine-rich protein 2 Hs.10526 43
IMAGE: 1715851 HBG2 Hemoglobin, gamma G Hs.302145 44 IMAGE: 155467
SLC9A3R2 Solute carrier family 9 Hs.440896 45 IMAGE: 561743 PPP1R1A
Protein phosphatase 1 Hs.435238 46 IMAGE: 565075 STC1 Stanniocalcin
1 Hs.25590 47 IMAGE: 1541958 POU2AF1 POU domain associating factor
Hs.2407 48 IMAGE: 324122 ESM1 Endothelial cell-specific molecule 1
Hs.129944 49 IMAGE: 80338 SELENBP1 Selenium binding protein 1
Hs.334841 50 IMAGE: 1472754 COX6B1 Cytochrome c oxidase
(ubiquitous) Hs.431668 51 IMAGE: 233583 IL1R2 Interleukin 1
receptor, type II Hs.25333 52 IMAGE: 490060 RNF159 Ring finger
protein (C3HC4 type) Hs.246914 53 IMAGE: 1185475 ABCC5 ATP-binding
cassette C Hs.22010 54 IMAGE: 120551 LPIN2 Lipin 2 Hs.437425 55
IMAGE: 162772 EGR1 Early growth response 1 Hs.326035 56 IMAGE:
322029 MAPK9 Mitogen-activated protein kinase 9 Hs.348446 57 IMAGE:
1305158 KIAA1219 KIAA1219 protein Hs.348929 58 IMAGE: 2505604 SCYE1
Endothelial monocyte-activating) Hs.105656 59 IMAGE: 1240813 IGKC
Immunoglobulin kappa constant Hs.377975 60 IMAGE: 257637 RRBP1
Ribosome binding protein 1 homolog Hs.98614 61 IMAGE: 381522 PP1057
Hypothetical protein PP1057 Hs.108557 62 IMAGE: 455123 MTSS1
Metastasis suppressor 1 Hs.77694
TABLE-US-00002 TABLE 2 Genes of known function in renal biopsies
whole blood predictive of graft loss following acute rejection.
Unigene Rank Clone Symbol Gene ID 1 IMAGE: 2134209 ZNF41 Zinc
finger protein 41 Hs.143700 2 IMAGE: 1241524 TCL1A T-cell
leukemia/lymphoma 1A Hs.2484 3 IMAGE: 704915 TAP1 Transporter 1
(MDR/TAP) Hs.352018 4 IMAGE: 267600 STAT6 Interleukin-4 induced
STAT6 Hs.437475 5 IMAGE: 26599 STAT1 Interleukin-4 induced STAT1
Hs.21486 6 IMAGE: 210405 PSME2 Proteasome activator Hs.434081 7
IMAGE: 1240661 PSMB9 Proteasome beta type, 9 Hs.381081 8 IMAGE:
705046 PML Promyelocytic leukemia Hs.89633 9 IMAGE: 824340 NCF1
Neutrophil cytosolic factor 1 Hs.1583 10 IMAGE: 753313 LAPTM5
Lysosomal-associated protein-5 Hs.436200 11 IMAGE: 1351990 ISG20
Interferon stimulated gene 20 kDa Hs.105434 12 IMAGE: 1672498 IGLV@
Ig lambda variable group Hs.449601 13 IMAGE: 1240590 IGLC2 Ig
lambda constant 2 Hs.405944 14 IMAGE: 1240813 IGKC Ig kappa
constant Hs.377975 15 IMAGE: 1604703 HLA-F MHC complex, class I, F
Hs.411958 16 IMAGE: 2448698 HLA-DRB6 MHC, class II, DR beta 6
(pseudogene) Hs.534338 17 IMAGE: 461769 HLA-DRB5 MHC complex, class
II, DR beta 5 Hs.308026 18 IMAGE: 1241341 HLA-DRB3 MHC complex,
class II, DR beta 3 Hs.520049 19 IMAGE: 1241211 HLA-DPB1 MHC
complex, class II, DP beta 1 Hs.368409 20 IMAGE: 203527 HLA-A MHC
complex, class I, A Hs.181244 21 IMAGE: 853906 HCG4P6 HLA complex
group 4 pseudogene 6 Hs.512759 22 IMAGE: 841008 GBP1 Guanylate
binding 1, interferon-inducible Hs.62661 23 IMAGE: 277522 DAF Decay
accelerating factor complement (CD55) Hs.408864 24 IMAGE: 269295
CD83 CD83 antigen (Activated B lymphocytes) Hs.444310 25 IMAGE:
276727 CD69 CD69 antigen (early T-cell activation antigen) Hs.82401
26 IMAGE: 200720 CD38 CD38 antigen (p45) Hs.174944 27 IMAGE:
2000918 CAS1 O-acetyltransferase Hs.324725 28 IMAGE: 67042 APOM
Apolipoprotein M Hs.247323 29 IMAGE: 488143 IGHM Immunoglobulin
heavy locus Hs.103995 30 IMAGE: 207718 TASS Ig light chain variable
region Hs.449578
In certain embodiments, at least one of the genes/proteins in the
prepared expression profile is a graft survival/rejection
indicative gene from Tables 1 and/or 2, where the expression
profile may include expression data for 5, 10, 20, 50, 75 or more
of, including all of, the genes/proteins listed in Tables 1 and/or
2. The number of different genes/proteins whose expression and/or
quantity data, i.e., presence or absence of expression, as well as
expression/quantity level, that are included in the expression
profile that is generated may vary, but may be at least 2, and in
certain embodiments ranges from 2 to about 100 or more, sometimes
from 3 to about 75 or more, including from about 4 to about 70 or
more.
In certain embodiments, additional genes beyond those listed in
Tables 1 and/or 2, may be assayed, such as genes whose expression
pattern can be used to evaluate additional transplant
characteristics, including but not limited to: acute rejection
(e.g., the genes identified as AR in Table 3, below); chronic
allograft injury (chronic rejection) in blood (e.g., the genes
identified as CR in Table 3, below); immunosuppressive drug
toxicity or adverse side effects including drug-induced
hypertension (e.g., the genes identified as DT in Table 3, below);
age or body mass index associated genes that correlate with renal
pathology or account for differences in recipient age-related graft
acceptance (e.g., the genes identified as BMI in Table 3, below);
immune tolerance markers in whole blood (e.g., the genes identified
as TOL in Table 3, below); genes found in literature surveys with
immune modulatory roles that may play a role in transplant outcomes
(e.g., the genes identified as Lit. in Table 3, below); as well as
other array assay function related genes, e.g., for assessing
sample quality (3'- to 5'-bias in probe location), sampling error
in biopsy-based studies, cell surface markers, and normalizing
genes for calibrating hybridization results (see e.g., the genes
identified as Contr. in Table 3, below); and the like.
A representative collection of genes that includes not only graft
survival/rejection genes of Tables 1 and 2 above, but also
additional graft characterizing genes (e.g., specific for DT, CAN,
and immune tolerance) is in Table 3.
TABLE-US-00003 TABLE 3 Genes of known function of prognostic value
compiled for a custom transplantation chip (TxChip VI). Symbol Name
mRNA Tissue Study ACOX1 Acyl-Coenzyme A oxidase 1, palmitoyl
NM_004035 Blood AR ADD3 Adducin 3 (gamma) NM_016824 Blood AR ADM
Adrenomedullin NM_001124 Blood AR AHR Aryl hydrocarbon receptor
NM_001621 Blood AR ATP1A1 ATPase, Na+/K+ transporting, alpha 1
NM_000701 Blood AR BUB1B BUB1 budding uninhibited by benzimidazoles
NM_001211 Blood AR CASP8 Caspase 8, apoptosis-related cysteine
protease NM_001228 Blood AR CASP8AP2 CASP8 associated protein 2
NM_012115 Blood AR CCNC Cyclin C NM_005190 Blood AR CD21 CD21
B-cell receptor for complement C3d0 Y00649 Blood AR CD69 CD69
antigen (early T-cell activation antigen) NM_001781 Blood AR CD8A
CD8 antigen, alpha polypeptide (p32) NM_001768 Blood AR CDIPT
Phosphatidylinositol synthase NM_145752 Blood AR COX6C Cytochrome c
oxidase subunit VIc NM_004374 Blood AR CSNK1A1 Casein kinase 1,
alpha 1 NM_001892 Blood AR DUSP1 Dual specificity phosphatase 1
NM_004417 Blood AR DUSP3 Dual specificity phosphatase 3 NM_004090
Blood AR EIF1A Eukaryotic translation initiation factor 1A
NM_001412 Blood AR EIF2S3 Eukaryotic translation initiation factor
2 NM_001415 Blood AR GNLY Granulysin NM_006433 Blood AR GOLGIN-67
Golgin-67 XM_496064 Blood AR AHSA2 Activator of heat shock ATPase
NM_152392 Blood AR HIST1H2BC Histone 1, H2bc NM_003526 Blood AR
IFNAR2 Interferon (alpha, beta and omega) receptor 2 NM_000874
Blood AR IGHG1 Ig heavy constant gamma 1 (G1m marker) AB067073
Blood AR IL1R2 Interleukin 1 receptor, type II NM_004633 Blood AR
MAPK1 Mitogen-activated protein kinase 1 NM_002745 Blood AR MIF
Macrophage migration inhibitory factor NM_002415 Blood AR SCYE1
Endothelial monocyte-activating NM_004757 Blood AR TGFBR3 TGFb
receptor III (betaglycan) NM_003243 Blood AR TM4SF9 Transmembrane 4
superfamily member 9 NM_005723 Blood AR IGHM Immunoglobulin heavy
constant mu X58529 Blood AR ISG20 Interferon stimulated gene 20 kDa
NM_002201 Blood AR KIAA1014 FNBP4 formin binding protein 4 AB023231
Blood AR LIV-1 SLC39A6 metal ion transporter NM_015359 Blood AR
MAPKAPK5 Mitogen-activated protein kinase NM_003668 Blood AR MDM4
p53 binding protein NM_002393 Blood AR MYT1 Myelin transcription
factor 1 NM_004535 Blood AR NAB1 EGR1 binding protein 1 NM_005966
Blood AR NFKB1 NFkB enhancer in B-cells 1 (p105) NM_003998 Blood AR
PC4 RNA polymerase II transcription cofactor 4 NM_006713 Blood AR
PKM2 Pyruvate kinase, muscle NM_002654 Blood AR PTP4A1 Protein
tyrosine phosphatase NM_003463 Blood AR RBL2 Retinoblastoma-like 2
(p130) NM_005611 Blood AR RBM3 RNA binding motif 3 (RNP1, RRM)
NM_006743 Blood AR REL V-rel viral oncogene homolog NM_002908 Blood
AR RPL22 Ribosomal protein L22 NM_000983 Blood AR RPS24 Ribosomal
protein S24 NM_033022 Blood AR RPS27 Ribosomal protein S27
NM_001030 Blood AR RPS4Y RPS4Y ribosomal protein S4 NM_001008 Blood
AR SATB1 Special AT-rich sequence binding protein NM_002971 Blood
AR SDS3 Likely ortholog of mouse Sds3 NM_022491 Blood AR SSBP1
Single-stranded DNA binding protein 1 NM_003143 Blood AR SSI-3
SOCS3 suppressor of cytokine signaling 3 NM_003955 Blood AR STK4
Serine/threonine kinase 4 NM_006282 Blood AR TBRG1 Transforming
growth factor beta regulator 1 NM_032811 Blood AR TCF7
Transcription factor 7 (T-cell specific) NM_201633 Blood AR TOP2B
Topoisomerase (DNA) II beta 180 kDa NM_001068 Blood AR TRIM T-cell
receptor interacting molecule NM_016388 Blood AR TRRAP
Transcription domain-associated protein NM_003496 Blood AR UBA52
Ubiquitin A-52-ribosomal protein fusion NM_003333 Blood AR UBB
Ubiquitin B NM_018955 Blood AR UBE2B Ubiquitin-conjugating enzyme
E2B NM_003337 Blood AR UBN1 Ubinuclein 1 NM_016936 Blood AR USP25
Ubiquitin specific protease 25 NM_013396 Blood AR AIM1 Absent in
melanoma 1 XM_166300 Biopsy AR CD38 CD38 antigen (p45) NM_001775
Biopsy AR CDS1 CDP-diacylglycerol synthase NM_001263 Biopsy AR
CSF1R Feline sarcoma viral (v-fms) homolog NM_005211 Biopsy AR DR1
Down-regulator of transcription 1 NM_001938 Biopsy AR FGL2
Fibrinogen-like 2 NM_006682 Biopsy AR FLJ13612 Calcium binding
protein AI635773 Biopsy AR HLA-A MHC class I, A NM_002116 Biopsy AR
HLA-B MHC class I, B NM_005514 Biopsy AR HLA-C MHC class I, C
NM_002117 Biopsy AR HLA-DPA1 MHC class II, DP alpha 1 NM_033554
Biopsy AR HLA-DRA MHC class II, DR alpha NM_019111 Biopsy AR IGKC
Ig kappa constant AB064140 Blood AR TNFSF10 TNF superfamily, member
10 NM_003810 Blood AR IGLJ3 IGLa Immunoglobulin lambda AI146764
Biopsy AR MYH10 Myosin, heavy polypeptide 10 NM_005964 Biopsy AR
NKTR Natural killer-tumor recognition sequence NM_005385 Biopsy AR
PAX8 Paired box gene 8 NM_013951 Biopsy AR POLR2B Polymerase (RNA)
II polypeptide B NM_000938 Biopsy AR RGN Regucalcin (senescence
marker protein-30) NM_004683 Biopsy AR SCNN1A Sodium channel,
nonvoltage-gated 1 alpha NM_001038 Biopsy AR SIM2 Single-minded
homolog 2 NM_009586 Biopsy AR TACSTD2 Calcium signal transducer 2
NM_002353 Biopsy AR VCAM1 Vascular cell adhesion molecule 1
NM_001078 Biopsy AR YARS Tyrosyl-tRNA synthetase NM_003680 Biopsy
AR ZFP36L1 Zinc finger protein 36 NM_004926 Biopsy AR HLA-DPB1 MHC,
class II, DP beta 1 NM_002121 Biopsy AR HLA-DRB3 MHC, class II, DR
beta 4 NM_022555 Biopsy AR ACK1 Cdc42-associated kinase 1 NM_005781
Biopsy AR HLA-F MHC, class I, F NM_018950 Biopsy AR ICAM5
Intercellular adhesion molecule 5 NM_003259 Biopsy AR REG1A
Regenerating islet-derived 1 alpha NM_002909 Biopsy AR GSTA2
Glutathione S-transferase A2 NM_000846 Biopsy AR HLA-DRB5 MHC class
II, DR beta 4 NM_002125 Biopsy AR HLA-DQA1 MHC class II, DQ alpha 1
NM_002122 Biopsy AR HLA-DQB1 MHC class II, DQ beta 1 NM_002123
Biopsy AR RFXANK Regulatory factor X-associated ankyrin NM_003721
Biopsy AR STAT6 Interleukin-4 induced STAT6 NM_003153 Biopsy AR
TAP1 Transporter 1 (MDR/TAP) NM_000593 Biopsy AR DAF Decay
accelerating factor (CD55) NM_000574 Biopsy AR CD83 CD83 antigen
(activated B lymphocytes) NM_004233 Biopsy AR STAT1 Interleukin-4
induced STAT1 NM_007315 Biopsy AR LTBR Lymphotoxin beta receptor
NM_002342 Biopsy AR KCNJ1 Potassium inwardly-rectifying channel
NM_000220 Biopsy AR SLPI Secretory leukocyte protease inhibitor
NM_003064 Biopsy AR CD34 CD34 antigen NM_001773 Biopsy AR HOXB5
Homeo box B5 NM_002147 Biopsy AR IL6R Interleukin 6 receptor
NM_181359 Biopsy AR DAPK1 Death-associated protein kinase 1
NM_004938 Biopsy AR HOXD9 Homeo box D9 NM_014213 Biopsy AR TCF21
Transcription factor 21 NM_003206 Biopsy AR MAL T-cell
differentiation protein NM_022438 Biopsy AR MAF V-maf fibrosarcoma
homolog NM_005360 Blood AR NCOR2 Nuclear receptor co-repressor 2
NM_006312 Blood CR ZFP106 Zinc finger protein 106 homolog NM_022473
Blood CR RPL23 Ribosomal protein L23 NM_000978 Blood CR CPVL
Carboxypeptidase, vitellogenic-like NM_019029 Blood CR ENO2 Enolase
2 (gamma, neuronal) NM_001975 Blood CR CAPN2 Calpain 2, (m/II)
large subunit NM_001748 Blood CR FGFR4 Fibroblast growth factor
receptor 4 NM_002011 Blood CR CD68 CD68 antigen NM_001251 Blood CR
HK3 Hexokinase 3 (white cell) NM_002115 Blood CR DUSP6 Dual
specificity phosphatase 6 NM_001946 Blood CR IL6ST Interleukin 6
signal transducer NM_002184 Blood CR LATS2 LATS, large tumor
suppressor 2 NM_014572 Blood CR MIC2 CD99 antigen NM_002414 Blood
CR MMP23B Matrix metalloproteinase 23B NM_006983 Blood CR ZNF511
Zinc finger protein 511 NM_145806 Blood CR ANXA5 Annexin A5
NM_001154 Blood CR ID2 Inhibitor of DNA binding 2 NM_002166 Blood
CR PRKRIR RNA dependent p58 repressor NM_004705 Blood CR SGK
Serum/glucocorticoid regulated kinase NM_005627 Blood CR S100A10
S100 calcium binding protein A10 NM_002966 Blood CR CYP51
Cytochrome P450, family 51A NM_000786 Blood CR ITGA4 Integrin,
alpha 4 (antigen CD49D) NM_000885 Blood CR ADAM10 A disintegrin and
metalloproteinase10 NM_001110 Blood CR HNRPK Nuclear
ribonucleoprotein K NM_031262 Blood CR ITGAV Integrin, alpha V
(CD51) NM_002210 Blood CR JUN V-jun sarcoma virus 17 homolog
NM_002228 Blood CR PRKAR2B Protein kinase regulator NM_002736 Blood
CR TIE Tyrosine kinase with Ig and EGF domains NM_005424 Blood CR
IQGAP2 GTPase activating protein 2 NM_006633 Blood CR MAP4K1
Mitogen-activated protein kinase 1 NM_007181 Blood CR ILF3
Interleukin enhancer binding factor 3 NM_012218 Blood CR SGKL
Serum/glucocorticoid regulated kinase-like NM_013257 Blood CR GLS
Glutaminase NM_014905 Blood CR DPYD Dihydropyrimidine dehydrogenase
NM_000110 Blood CR ACADM Acyl-Coenzyme A dehydrogenase NM_000016
Biopsy DT AUTS2 Autism susceptibility candidate 2 NM_015570 Biopsy
DT CA2 Carbonic anhydrase II NM_000067 Biopsy DT CTNNA1 Catenin
(cadherin-associated protein) NM_001903 Biopsy DT CXCL12 Stromal
cell-derived factor 1 NM_000609 Biopsy DT DDR1 Discoidin domain
receptor family, member 1 NM_013994 Biopsy DT DECR1 2,4-dienoyl CoA
reductase 1, mitochondrial NM_001359 Biopsy DT DEDD Death effector
domain containing NM_032998 Biopsy DT DPP4 Dipeptidylpeptidase 4
(CD26) NM_001935 Biopsy DT ITM2B Integral membrane protein 2B
NM_021999 Biopsy DT KIAA0436 L-type neutral amino acid transporter
AB007896 Biopsy DT LDHB Lactate dehydrogenase B NM_002300 Biopsy DT
LEPR Leptin receptor NM_002303 Biopsy DT LRBA LPS-responsive
vesicle trafficking NM_006726 Biopsy DT MUT Methylmalonyl Coenzyme
A mutase NM_000255 Biopsy DT NAT1 N-acetyltransferase 1 NM_000662
Biopsy DT NAT2 N-acetyltransferase 2 NM_000015 Biopsy DT NUP50
Nucleoporin 50 kDa NM_153645 Biopsy DT PAFAH1B1 Platelet-activating
factor NM_000430 Biopsy DT PDZK3 PDZ domain containing 3 NM_178140
Biopsy DT PLCL2 Phospholipase C-like 2 NM_015184 Biopsy DT PPP2CB
Protein phosphatase 2 NM_004156 Biopsy DT PRKCM Protein kinase C,
mu NM_002742 Biopsy DT PTPN3 Protein tyrosine phosphatase NM_002829
Biopsy DT REST RE1-silencing transcription factor NM_005612 Biopsy
DT SGCB Sarcoglycan, beta NM_000232 Biopsy DT SHB Src homology 2
domain containing NM_003028 Biopsy DT SORL1 Sortilin-related
receptor, L NM_003105 Biopsy DT SULT1E1 Sulfotransferase family 1E
NM_005420 Biopsy DT CBL Cas-Br-Transforming sequence NM_005188
Biopsy DT CXCL1 Chemokine (C--X--C motif) ligand 1 NM_001511 Biopsy
DT FGF2 Fibroblast growth factor 2 (basic) NM_002006 Biopsy DT
GPRK5 G protein-coupled receptor kinase 5 NM_005308 Biopsy DT ITSN2
Intersectin 2 NM_006277 Biopsy DT BCL2L13 BCL2-like 13 (apoptosis
facilitator) AA279535 Biopsy BMI BDKRB2 Bradykinin receptor B2
NM_000623 Biopsy BMI DDX3 DEAD/H (Asp-Glu-Ala-Asp/His) box 3
NM_001356 Biopsy BMI FOXM1 Forkhead box M1 NM_021953 Biopsy BMI
HMOX2 Heme oxygenase (decycling) 2 NM_002134 Biopsy BMI IFNGR1
Interferon gamma receptor 1 NM_000416 Biopsy BMI IGFBP1
Insulin-like growth factor binding protein 1 NM_000596 Biopsy BMI
IGFBP5 Insulin-like growth factor binding protein 5 NM_000599
Biopsy BMI LRP2 Low density lipoprotein-related protein 2 NM_004525
Biopsy BMI MCM7 Minichromosome maintenance deficient 7 NM_182776
Biopsy BMI NPPB Natriuretic peptide precursor B NM_002521 Biopsy
BMI NPR1 Natriuretic peptide receptor A NM_000906 Biopsy BMI
PAXIP1L PAX transcription activation interacting NM_007349 Biopsy
BMI PDCD5 Programmed cell death 5 NM_004708 Biopsy BMI RBX1
Ring-box 1 NM_014248 Biopsy BMI RPL27 Ribosomal protein L27
NM_000988 Biopsy BMI SBA2 WD repeat and SOCS box containing protein
AA043793 Biopsy BMI SERPINB6 Proteinase inhibitor, clade B
(ovalbumin) NM_004568 Biopsy BMI SLC22A5 Solute carrier family 22
NM_003060 Biopsy BMI SLC38A2 Solute carrier family 38, member 2
NM_018976 Biopsy BMI SMT3H2 Suppressor of MIF NM_006937 Biopsy BMI
TJP4 Tight junction protein 4 (peripheral) NM_080604 Biopsy BMI
TP53INP1 p53 inducible nuclear protein 1 NM_033285 Biopsy BMI
BHLHB2 Basic helix-loop-helix domain containing NM_003670 Biopsy
BMI CSPG2 Chondroitin sulfate proteoglycan 2 NM_004385 Biopsy BMI
GPD1 Glycerol-3-phosphate dehydrogenase 1 NM_005276 Biopsy BMI
GTPBP4 GTP binding 4; Chronic renal failure gene NM_012341 Biopsy
BMI HIF1A Hypoxia-inducible factor 1, alpha NM_001530 Biopsy BMI
MMP7 Matrix metalloproteinase 7 NM_002423 Biopsy BMI SLC2A3
Facilitated glucose transporter NM_006931 Biopsy BMI THBS1
Thrombospondin 1 NM_003246 Biopsy BMI TNC Tenascin C (hexabrachion)
NM_002160 Biopsy BMI HLA-G HLA-G histocompatibility antigen, class
I, G NM_002127 Blood TOL IGHG3 Ig heavy constant gamma 3 AK097306
Blood TOL BUR1 Budding uninhibited (cell cycle regulator) NM_004336
Blood TOL CCNB2 Cyclin B2 NM_004701 Blood TOL TACSTD1
Tumor-associated calcium signaling NM_002354 Blood TOL DHRS2
Dehydrogenase/reductase (SDR family) AK092834 Blood TOL BMP7 Bone
morphogenetic protein 7 NM_001719 Blood TOL AKR1C1 Aldo-keto
reductase family 1C1 NM_001353 Blood TOL B4GALT2 UDP-Gal
1,4-galactosyltransferase NM_003780 Blood TOL TCEB3 Transcription
elongation factor B (SIII) NM_003198 Blood TOL MLPH Melanophilin
NM_024101 Blood TOL SERPINH2 Heat shock protein 47 (proteinase
inhibitor) NM_001235 Blood TOL RRM2 Ribonucleotide reductase M2
polypeptide NM_001034 Blood TOL SERPINA3 Alpha-1 antiproteinase,
antitrypsin NM_001085 Blood TOL SERPINA5 Alpha-1 antiproteinase,
antitrypsin NM_000624 Blood TOL CTNNAL1 Catenin
(cadherin-associated protein) NM_003798 Blood TOL SPARC Secreted
protein, cysteine-rich (osteonectin) NM_003118 Blood TOL C1S C1S
complement component 1 NM_001734 Blood TOL SRPUL SRPUL sushi-repeat
protein NM_006307 Blood TOL MMP2 Matrix metalloproteinase 2
NM_004530 Blood TOL SLC7A7 Cationic amino acid transporter
NM_003982 Blood TOL EPOR Erythropoietin receptor NM_000121 Blood
TOL PRAME Preferentially expressed antigen in melanoma NM_006115
Blood TOL AFP Alpha-fetoprotein NM_001134 Blood TOL MAPK9
Mitogen-activated protein kinase 9 NM_002752 Blood TOL
NR2F2 Nuclear receptor subfamily 2F2 NM_021005 Blood TOL PFN2
Profilin 2 NM_053024 Blood TOL SLC38A6 Solute carrier family 38,
member 6 BC050349 Blood TOL MYOM2 Myomesin (M-protein) 2, 165 kDa
NM_003970 Blood TOL RBP1 Retinol binding protein 1, cellular
NM_002899 Blood TOL TK1 Thymidine kinase 1, soluble NM_003258 Blood
TOL IFITM3 Interferon induced transmembrane protein 3 NM_021034
Blood TOL APOH Apolipoprotein H (beta-2-glycoprotein I) NM_000042
Blood TOL EVI2A Ecotropic viral integration site 2A NM_014210 Blood
TOL CD9 CD9 antigen (p24) NM_001769 Blood TOL NKG7 Natural killer
cell group 7 sequence NM_005601 Blood TOL CDKN3 Cyclin-dependent
kinase inhibitor 3 NM_005192 Blood TOL TCL1A T-cell
leukemia/lymphoma 1A NM_021966 Blood TOL PYCR1
Pyrroline-5-carboxylate reductase 1 NM_153824 Blood TOL TM4SF5
Transmembrane 4 superfamily member 5 NM_003963 Blood TOL GAGEB1 G
antigen, family B, 1 (prostate associated) NM_003785 Blood TOL PCP4
Purkinje cell protein 4 NM_006198 Blood TOL LGMN Legumain NM_005606
Blood TOL PIR Pirin (iron-binding nuclear protein) NM_178238 Blood
TOL PAICS Phosphoribosylaminoimidazole carboxylase NM_006452 Blood
TOL IGFBP3 Insulin-like growth factor binding protein 3 NM_000598
Blood TOL PSMB9 Proteasome subunit NM_002800 Blood TOL N33 Putative
prostate cancer tumor suppressor NM_006765 Blood TOL DP1 Polyposis
locus protein 1 (DP1) NM_005669 Blood TOL TFDP1 Transcription
factor Dp-1 NM_007111 Blood TOL OSF-2 OSF-2 osteoblast specific
factor 2 NM_000358 Blood TOL COL3A1 Collagen, type III, alpha 1
NM_000090 Blood TOL TIMP3 TIMP3 tissue inhibitor of
metalloproteinase 3 NM_000362 Blood TOL SPP1 Osteopontin, early
T-lymphocyte activation 1 NM_000582 Blood TOL NQO1 NQO1 NAD(P)H
dehydrogenase NM_000903 Blood TOL TOP2A Topoisomerase (DNA) II
alpha 170 kDa NM_001067 Blood TOL CCND2 Cyclin D2 NM_001759 Blood
TOL CNN3 CNN3 calponin 3, acidic AI969128 NM_001839 Blood TOL
COL6A1 Collagen, type VI, alpha 1 NM_001848 Blood TOL CTGF
Connective tissue growth factor NM_001901 Blood TOL EGR1 Early
growth response 1 (EGR1) NM_001964 Blood TOL SDC2 Syndecan 2
NM_002998 Blood TOL TCF3 Transcription factor 3 NM_003200 Blood TOL
TFAP2C Transcription factor AP-2 gamma NM_003222 Blood TOL NRP1
Neuropilin 1 NM_003873 Blood TOL GITR TNF receptor superfamily18
(TNFRSF18) NM_004195 Blood TOL COL6A3 Collagen, type VI, alpha 3
NM_004369 Blood TOL EPHA2 EPHA2 EphA2 NM_004431 Blood TOL PDE1A
ARHE ras homolog gene family NM_005168 Blood TOL FAT Tumor
suppressor homolog 1 NM_005245 Blood TOL KIFC3 Kinesin family
member C3 NM_005550 Blood TOL NR2F1 Nuclear receptor subfamily 2F1
NM_005654 Blood TOL CAP2 CAP, adenylate cyclase-associated 2
NM_006366 Blood TOL BACE2 Beta-site APP-cleaving enzyme 2 NM_012105
Blood TOL FADS1 Fatty acid desaturase 1 NM_013402 Blood TOL MELK
Maternal embryonic leucine zipper kinase NM_014791 Blood TOL DKK3
Dickkopf homolog 3 (Xenopus laevis) NM_015881 Blood TOL CCNB1
Cyclin B1 NM_031966 Blood TOL CALD1 Caldesmon 1 NM_033138 Blood TOL
CASP1 Caspase 1, (interleukin 1b convertase) NM_033292 Blood TOL
KNSL5 Kinesin-like 5 (mitotic kinesin-like protein 1) NM_138555
Blood TOL STK6 Serine/threonine kinase 6 NM_198433 Blood TOL CD59
CD59 antigen p18-20 NM_203330 Blood TOL FN1 Fibronectin 1 NM_212482
Blood TOL SERPINE2 Serine proteinase inhibitor NM_006216 Blood TOL
CDH2 Cadherin 2, type 1, N-cadherin NM_001792 Blood TOL CCNE1
Cyclin E1 NM_001238 Blood TOL SEMA3F Ig short basic domain,
secreted NM_004186 Blood TOL MAD2L1 MAD2 mitotic arrest
deficient-like 1 NM_002358 Blood TOL CYR61 Cysteine-rich,
angiogenic inducer, 61 NM_001554 Blood TOL TNFRSF7 CD27 TNF
receptor superfamily 7 NM_001242 Blood TOL FOXP3 Forkhead box P3
(FOXP3), mRNA NM_014009 Blood TOL ABCA4 ATP-binding cassette,
sub-family A (ABC1) NM_000350 Biopsy Control HNK-1 HNK-1
sulfotransferase AF033827 Biopsy Control UCP2 Uncoupling protein 2
NM_003355 Biopsy Control DAB2 Mitogen-responsive phosphoprotein
NM_001343 Biopsy Control AQP3 Aquaporin 3 NM_004925 Biopsy Control
CRABP1 Cellular retinoic acid binding protein 1 NM_004378 Biopsy
Control KCNAB2 Potassium voltage-gated channel NM_003636 Biopsy
Control TNNT2 Troponin T2, cardiac NM_000364 Biopsy Control APP
Amyloid beta (A4) precursor protein NM_000484 Biopsy Control FABP3
Fatty acid binding protein 3 NM_004102 Biopsy Control PODXL
Podocalyxin-like NM_005397 Biopsy Control ALPI Alkaline
phosphatase, intestinal NM_001631 Biopsy Control MAPT
Microtubule-associated protein tau NM_005910 Biopsy Control KHK
Ketohexokinase (fructokinase) NM_000221 Biopsy Control 18S 18s
ribosomal RNA M10098 All Control ACTB Actin, beta NM_001101 All
Control GAPD Glyceraldehyde-3-phosphate dehydrogenase NM_002046 All
Control GSUSB Glucuronidase, beta NM_000181 All Control HPRT1
Hypoxanthine phosphoribosyltransferase 1 NM_000194 All Control
SCYA3 Chemokine (C--C motif) ligand 3 NM_002983 All Control LMO2
LIM domain only 2 (LMO2) NM_005574 All Control BCL6 B-cell
CLL/lymphoma 6 NM_001706 All Control IkB2 NFkB enhancer in B-cells
inhibitor NM_020529 All Control APC Adenomatosis polyposis coli
NM_000038 All Control BAG2 BCL2-associated athanogene 2 (BAG2)
NM_004282 All Control CREBBP CREB binding protein NM_004380 All
Control KLRB1 Killer cell lectin-like receptor B1 NM_002258 All
Control TRADD TNFRSF1A-associated via death domain NM_003789 All
Control CXCL14 Chemokine (C--X--C motif) ligand 14 NM_004887 All
Control IL1A Interleukin 1, alpha NM_000575 All Control MMP1 Matrix
metalloproteinase 1 NM_002421 All Control MMP9 Matrix
metalloproteinase 9 NM_004994 All Control VEGFC Vascular
endothelial growth factor C NM_005429 All Control CD8A CD8 antigen,
alpha polypeptide (p32) NM_171827 Blood Control CD3G CD3G antigen,
gamma (TiT3 complex) NM_000073 Blood Control CD44 CD44 antigen
NM_000610 Blood Control CD4 CD4 antigen (p55) NM_000616 Blood
Control CD3D CD3D antigen, delta (TiT3 complex) NM_000732 Blood
Control CD3E CD3E antigen, epsilon (TiT3 complex) NM_000733 Blood
Control CD3Z CD3Z antigen, zeta (TiT3 complex) NM_000734 Blood
Control CD19 CD19 antigen NM_001770 Blood Control B220 Protein
tyrosine phosphatase receptor NM_002838 Blood Control CD138 CD138
syndecan 1 (SDC1) NM_002997 Blood Control CD43 Sialophorin (CD43)
NM_003123 Blood Control CD8B1 CD8 antigen, beta polypeptide 1 (p37)
NM_004931 Blood Control API5 Apoptosis inhibitor 5 NM_006595 All
Lit. Axin1 Axin 1 NM_003502 All Lit. Axin2 Axin 2 (conductin, axil)
NM_004655 All Lit. BAD BCL2-antagonist of cell death NM_032989 All
Lit. BIK BCL2-interacting killer (apoptosis-inducing) NM_001197 All
Lit. BMP4 Bone morphogenetic protein 4 NM_001202 All Lit. BTG1
B-cell translocation gene 1 NM_001731 All Lit. CASP10 Caspase 10,
apoptosis-related cysteine protease NM_001230 All Lit. CASP3
Caspase 3, apoptosis-related cysteine protease NM_004346 All Lit.
CASP4 Caspase 4, apoptosis-related cysteine protease NM_001225 All
Lit. CASP7 Caspase 7, apoptosis-related cysteine protease NM_001227
All Lit. CASP9 Caspase 9, apoptosis-related cysteine protease
NM_001229 All Lit. CCL18 Chemokine (C--C motif) ligand 18 NM_002988
All Lit. CD161 Killer cell lectin-like receptor B1 BC027885 All
Lit. CD20 Membrane-spanning 4A1 NM_152866 All Lit. CD22 CD22
antigen NM_001771 All Lit. CD48 CD48 antigen (B-cell membrane
protein) NM_001778 All Lit. CD80 CD80 antigen (B7-1 antigen)
NM_005191 All Lit. CDA08 T-cell immunomodulatory protein NM_030790
All Lit. CDC2 Cell division cycle 2, G1 to S and G2 to M NM_001786
All Lit. CDw108 Semaphorin Ig and GPI membrane anchor 7A, NM_003612
All Lit. CDW52 CDW52 antigen (CAMPATH-1 antigen) NM_001803 All Lit.
CIS4 STAT induced STAT inhibitor-4 NM_004232 All Lit. CTLA4
Cytotoxic T-lymphocyte-associated protein 4 NM_005214 All Lit. DAD1
Defender against cell death 1 NM_001344 All Lit. DAP3 Death
associated protein 3 NM_033657 All Lit. DAPK2 Death-associated
protein kinase 2 NM_014326 All Lit. DAPK3 Death-associated protein
kinase 3 NM_001348 All Lit. DAXX Death-associated protein 6
NM_001350 All Lit. EBF Early B-cell factor NM_024007 All Lit.
FCGR1A Fc fragment of IgG (receptor for CD64) NM_000566 All Lit.
GADD45B Growth arrest and DNA-damage-inducible NM_015675 All Lit.
GSR Glutathione reductase NM_000637 All Lit. GZMA Granzyme A
NM_006144 All Lit. GZMB Granzyme B NM_004131 All Lit. Gzmc Granzyme
C M18459 All Lit. GZMK Granzyme K NM_002104 All Lit. HLA-E MHC
class I, E NM_005516 All Lit. ICAM1 Intercellular adhesion molecule
1 (CD54) NM_000201 All Lit. ICAM3 Intercellular adhesion molecule 3
NM_002162 All Lit. IFI16 Interferon, gamma-inducible protein 16
NM_005531 All Lit. IFI35 Interferon-induced protein 35 NM_005533
All Lit. IFNG Interferon, gamma NM_000619 All Lit. IGBP1 Ig (CD79A)
binding protein 1 NM_001551 All Lit. IGJ Ig J polypeptide, linker
protein NM_144646 All Lit. IK IK cytokine, down-regulator of HLA II
NM_006083 All Lit. IL2RA Interleukin 2 receptor, alpha NM_000417
All Lit. IL4R Interleukin 4 receptor NM_000418 All Lit. IL6
Interleukin 6 (interferon, beta 2) NM_000600 All Lit. IL7R
Interleukin 7 receptor NM_002185 All Lit. IL8RB Interleukin 8
receptor, beta NM_001557 All Lit. IRF1 Interferon regulatory factor
1 NM_002198 All Lit. ITGAE Integrin, alpha E (CD103) NM_002208 All
Lit. JAK1 Janus kinase 1 NM_002227 All Lit. JAK2 Janus kinase 2
NM_004972 All Lit. MADH2 SMAD, mothers against DPP NM_005901 All
Lit. MAPK3 Mitogen-activated protein kinase 3 NM_002746 All Lit.
MDM2 p53 binding protein NM_002392 All Lit. MHC2TA MHC class II
transactivator NM_000246 All Lit. NK4 Natural killer cell
transcript 4 NM_004221 All Lit. NMI N-myc (and STAT) interactor
NM_004688 All Lit. PCNA Proliferating cell nuclear antigen
NM_002592 All Lit. PDCD2 Programmed cell death 2 NM_002598 All Lit.
PDCD7 Programmed cell death 7 NM_005707 All Lit. PDCD8 Programmed
cell death 8 NM_004208 All Lit. PDGFRB Platelet-derived growth
factor receptor NM_002609 All Lit. RhoA Ras homolog gene family,
member A NM_001664 All Lit. SIMRP7 Multidrug resistance-associated
protein 7 NM_033450 All Lit. SOD2 Superoxide dismutase 2,
mitochondrial NM_000636 All Lit. SSI-1 suppressor of cytokine
signaling 1 NM_003745 All Lit. STAT2 Signal transducer2, 113 kDa
NM_005419 All Lit. STAT3 Signal transducer 3 (acute-phase response
factor) NM_139276 All Lit. STAT4 Signal transducer 4 NM_003151 All
Lit. STAT5A Signal transducer 5A NM_003152 All Lit. STAT5B Signal
transducer a5B NM_012448 All Lit. STK21 Rho-interacting NM_007174
All Lit. TA-LRRP TNF receptor-associated factor 6 NM_145803 All
Lit. TCRA T-cell receptor active alpha-chain M12423 All Lit. TCRB T
cell receptor beta locus X60096 All Lit. TCRD T-cell receptor delta
chain (VJC-region) M21624 All Lit. TCRG T cell receptor gamma locus
X06774 All Lit. TFRC Transferrin receptor (p90, CD71) NM_003234 All
Lit. TGFA Transforming growth factor, alpha NM_003236 All Lit.
TGFB2 Transforming growth factor, beta 2 NM_003238 All Lit. THBS2
Thrombospondin 2 NM_003247 All Lit. TIA1 Cytotoxic
granule-associated RNA binding NM_022173 All Lit. TIEG2 TGFB
inducible early growth response 2 NM_003597 All Lit. TLR5 Toll-like
receptor 5 NM_003268 All Lit. TNFRSF1A TNF receptor superfamily,
member 1A NM_001065 All Lit. TNFRSF1B TNF receptor superfamily,
member 1B NM_001066 All Lit. TNFSF7 TNF (ligand) superfamily,
member 7 NM_001252 All Lit. TP53BP1 Tumor protein p53 binding
protein, 1 NM_005657 All Lit. TP53BP2 Tumor protein p53 binding
protein, 2 NM_005426 All Lit. TRAF1 TNF receptor-associated factor
1 NM_005658 All Lit. TRAF2 TNF receptor-associated factor 2
NM_021138 All Lit. TRAF3 TNF receptor-associated factor 3 NM_003300
All Lit. TRAF4 TNF receptor-associated factor 4 NM_004295 All Lit.
TRAP1 TNF receptor-associated protein 1 NM_004257 All Lit. TTK TTK
protein kinase NM_003318 All Lit. UBE1L Ubiquitin-activating enzyme
E1-like NM_003335 All Lit. VPREB3 Pre-B lymphocyte gene 3 NM_013378
All Lit. WNT1 MMTV integration site (WNT1) NM_005430 All Lit. ACE1
Ig receptor (PIGR) IgA nephritis NM_002644 All Lit. BAX
BCL2-associated X protein NM_138763 All Lit. BCL2 B-cell
CLL/lymphoma 2 NM_000633 All Lit. C3 Complement component 3
NM_000064 All Lit. CD28 CD28 antigen (Tp44) NM_006139 All Lit. CD86
CD86 antigen (B7-2 antigen) NM_006889 All Lit. ICOS Inducible
T-cell co-stimulator NM_012092 All Lit. IL10 Interleukin 10
NM_000572 All Lit. IL15 Interleukin 15 NM_000585 All Lit. IL2
Interleukin 2 NM_000586 All Lit. IL4 Interleukin 4 NM_000589 All
Lit. IL7 Interleukin 7 NM_000880 All Lit. IL8 Interleukin 8
NM_000584 All Lit. PRF1 Perforin 1 (pore forming protein) NM_005041
All Lit. RANTES Chemokine (C--C motif) ligand 5 (CCL5) NM_002985
All Lit. TBET Th1-specific T-box transcription factor NM_013351 All
Lit. TGFB1 TGF beta 1 NM_000660 All Lit. TNF TNF superfamily,
member 2 NM_000594 All Lit. TNFB Lymphotoxin alpha (TNF1 or LTA)
NM_000595 All Lit. TNFRSF5 CD40 TNF receptor superfamily 5
NM_001250 All Lit. TNFRSF6 CD95 = Fas TNF receptor superfamily 6
NM_000043 All Lit. VEGF Vascular endothelial growth factor
NM_003376 All Lit.
In certain embodiments, a collection of genes from Table 3 is
assayed, where in these embodiments the number of genes from Table
3 may be at least about 5%, at least about 10%, at least about 25%,
at least about 50%, at least about 75%, at least about 90% or more,
including all of the genes from Table 3.
In certain embodiments, the expression profile obtained is a
genomic or nucleic acid expression profile, where the amount or
level of one or more nucleic acids in the sample is determined,
e.g., the nucleic acid transcript of the gene of interest. In these
embodiments, the sample that is assayed to generate the expression
profile employed in the diagnostic methods is one that is a nucleic
acid sample. The nucleic acid sample includes a plurality or
population of distinct nucleic acids that includes the expression
information of the phenotype determinative genes of interest of the
cell or tissue being diagnosed. The nucleic acid may include RNA or
DNA nucleic acids, e.g., mRNA, cRNA, cDNA etc., so long as the
sample retains the expression information of the host cell or
tissue from which it is obtained. The sample may be prepared in a
number of different ways, as is known in the art, e.g., by mRNA
isolation from a cell, where the isolated mRNA is used as is,
amplified, employed to prepare cDNA, cRNA, etc., as is known in the
differential expression art. In certain embodiments, the sample is
prepared from a cell or tissue harvested from a subject to be
diagnosed, e.g., via biopsy of tissue, using standard protocols,
where cell types or tissues from which such nucleic acids may be
generated include any tissue in which the expression pattern of the
to be determined phenotype exists, including, but not limited to,
peripheral blood lymphocyte cells, etc, as reviewed above.
The expression profile may be generated from the initial nucleic
acid sample using any convenient protocol. While a variety of
different manners of generating expression profiles are known, such
as those employed in the field of differential gene expression
analysis, one representative and convenient type of protocol for
generating expression profiles is array-based gene expression
profile generation protocols. In certain embodiments, such
applications are hybridization assays in which a nucleic acid array
that displays "probe" nucleic acids for each of the genes to be
assayed/profiled in the profile to be generated is employed. In
these assays, a sample of target nucleic acids is first prepared
from the initial nucleic acid sample being assayed, where
preparation may include labeling of the target nucleic acids with a
label, e.g., a member of signal producing system. Following target
nucleic acid sample preparation, the sample is contacted with the
array under hybridization conditions, whereby complexes are formed
between target nucleic acids that are complementary to probe
sequences attached to the array surface. The presence of hybridized
complexes is then detected, either qualitatively or quantitatively.
Specific hybridization technology which may be practiced to
generate the expression profiles employed in the subject methods
includes the technology described in U.S. Pat. Nos. 5,143,854;
5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980;
5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992;
the disclosures of which are herein incorporated by reference; as
well as WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373
203; and EP 785 280. In these methods, an array of "probe" nucleic
acids that includes a probe for each of the phenotype determinative
genes whose expression is being assayed is contacted with target
nucleic acids as described above. Contact is carried out under
hybridization conditions, e.g., stringent hybridization conditions,
and unbound nucleic acid is then removed.
The resultant pattern of hybridized nucleic acid provides
information regarding expression for each of the genes that have
been probed, where the expression information is in terms of
whether or not the gene is expressed and, typically, at what level,
where the expression data, i.e., expression profile (e.g., in the
form of a transcriptosome), may be both qualitative and
quantitative.
Alternatively, non-array based methods for quantitating the levels
of one or more nucleic acids in a sample may be employed, including
quantitative PCR, and the like.
Where the expression profile is a protein expression profile, any
convenient protein quantitation protocol may be employed, where the
levels of one or more proteins in the assayed sample are
determined. Representative methods include, but are not limited to:
proteomic arrays, flow cytometry, standard immunoassays (e.g.,
ELISA assays), protein activity assays, including multiplex protein
activity assays, etc.
Following obtainment of the expression profile from the sample
being assayed, the expression profile is compared with a reference
or control profile to determine the particular graft
tolerant/intolerant phenotype of the cell or tissue, and therefore
host, from which the sample was obtained/derived. The terms
"reference" and "control" as used herein mean a standardized
pattern of gene expression or levels of expression of certain genes
to be used to interpret the expression signature of a given patient
and assign a graft tolerant/intolerant phenotype thereto. The
reference or control profile may be a profile that is obtained from
a cell/tissue known to have the desired phenotype, e.g., tolerant
phenotype, and therefore may be a positive reference or control
profile. In addition, the reference/control profile may be from a
cell/tissue known to not have the desired phenotype, e.g., an
intolerant phenotype, and therefore be a negative reference/control
profile.
In certain embodiments, the obtained expression profile is compared
to a single reference/control profile to obtain information
regarding the phenotype of the cell/tissue being assayed. In yet
other embodiments, the obtained expression profile is compared to
two or more different reference/control profiles to obtain more in
depth information regarding the phenotype of the assayed
cell/tissue. For example, the obtained expression profile may be
compared to a positive and negative reference profile to obtain
confirmed information regarding whether the cell/tissue has the
phenotype of interest.
The comparison of the obtained expression profile and the one or
more reference/control profiles may be performed using any
convenient methodology, where a variety of methodologies are known
to those of skill in the array art, e.g., by comparing digital
images of the expression profiles, by comparing databases of
expression data, etc. Patents describing ways of comparing
expression profiles include, but are not limited to, U.S. Pat. Nos.
6,308,170 and 6,228,575, the disclosures of which are herein
incorporated by reference. Methods of comparing expression profiles
are also described above.
The comparison step results in information regarding how similar or
dissimilar the obtained expression profile is to the
control/reference profile(s), which similarity/dissimilarity
information is employed to determine the phenotype of the
cell/tissue being assayed and thereby evaluate graft survival in
the subject. For example, similarity with a positive control
indicates that the assayed cell/tissue has a graft survival
phenotype. Likewise, similarity with a negative control indicates
that the assayed cell/tissue has a graft loss phenotype.
Depending on the type and nature of the reference/control
profile(s) to which the obtained expression profile is compared,
the above comparison step yields a variety of different types of
information regarding the cell/tissue that is assayed. As such, the
above comparison step can yield a positive/negative determination
of a graft survival phenotype of an assayed cell/tissue. In many
embodiments, the above-obtained information about the cell/tissue
being assayed is employed to diagnose a host, subject or patient
with respect to graft survival, as described above. In certain
embodiments, the determination/prediction of graft survival and
loss can be coupled with a determination of additional
characteristics of the graft and function thereof. For example, in
certain embodiments one can predict not only whether graft loss
will occur, but the mechanism of graft loss, e.g., via CAN or DT.
The first 9 genes in the cluster illustrated in FIG. 4 are
highly-differentially expressed between CAN and DT. As such,
evaluating one or more of these genes permits these two overlapping
conditions to be readily distinguished, such that one can readily
determine the presence of CAN or DT.
The subject methods further find use in pharmacogenomic
applications. In these applications, a subject/host/patient is
first diagnosed for graft function according to the subject
invention, and then treated using a protocol determined, at least
in part, on the results of the diagnosis. For example, a host may
be evaluated for the presence of absence of the graft survival
phenotype using a protocol such as the diagnostic protocol
described in the preceding section. The subject may then be treated
using a protocol whose suitability is determined using the results
of the diagnosis step. In embodiments, where the host is evaluated
for the presence or absence of CAN or DT, treatment protocols may
correspondingly be adjusted based on the obtained results. For
example, where the subject methods are employed to determine the
presence of CAN, immunosuppressive therapy can be modulated, e.g.,
increased or drugs changed, as is known in the art for the
treatment of CAN. Likewise, where the subject methods are employed
and detect the presence of DT, the immunosuppressive therapy can be
reduced in order to treat the DT. In practicing the subject
methods, a subject is typically screened for the presence of a
graft survival or loss phenotype following receipt of a graft or
transplant. The subject may be screened once or serially following
transplant receipt, e.g., weekly, monthly, bimonthly, half-yearly,
yearly, etc. In certain embodiments, the subject is screened
following occurrence of acute rejection (AR). In such embodiments,
the methods are employed to evaluate, e.g., predict, ultimate graft
loss or survival in the subject following AR.
The subject methods may be employed with a variety of different
types of transplant subjects. In many embodiments, the subjects are
within the class mammalian, including the orders carnivore (e.g.,
dogs and cats), rodentia (e.g., mice, guinea pigs, and rats),
lagomorpha (e.g. rabbits) and primates (e.g., humans, chimpanzees,
and monkeys). In certain embodiments, the animals or hosts, i.e.,
subjects (also referred to herein as patients) will be humans.
The methods may be used to evaluate survival of a variety of
different types of grafts. Grafts of interest include, but are not
limited to: transplanted heart, kidney, lung, liver, pancreas,
pancreatic islets, brain tissue, stomach, large intestine, small
intestine, cornea, skin, trachea, bone, bone marrow, muscle,
bladder or parts thereof.
Databases of Expression Profiles of Phenotype Determinative
Genes
Also provided are databases of expression profiles of graft
survival and/or graft loss phenotype determinative genes. Such
databases will typically comprise expression profiles of various
cells/tissues having graft tolerant phenotypes, negative expression
profiles, etc., where such profiles are further described
below.
The expression profiles and databases thereof may be provided in a
variety of media to facilitate their use. "Media" refers to a
manufacture that contains the expression profile information of the
present invention. The databases of the present invention can be
recorded on computer readable media, e.g. any medium that can be
read and accessed directly by a computer. Such media include, but
are not limited to: magnetic storage media, such as floppy discs,
hard disc storage medium, and magnetic tape; optical storage media
such as CD-ROM; electrical storage media such as RAM and ROM; and
hybrids of these categories such as magnetic/optical storage media.
One of skill in the art can readily appreciate how any of the
presently known computer readable mediums can be used to create a
manufacture comprising a recording of the present database
information. "Recorded" refers to a process for storing information
on computer readable medium, using any such methods as known in the
art. Any convenient data storage structure may be chosen, based on
the means used to access the stored information. A variety of data
processor programs and formats can be used for storage, e.g. word
processing text file, database format, etc.
As used herein, "a computer-based system" refers to the hardware
means, software means, and data storage means used to analyze the
information of the present invention. The minimum hardware of the
computer-based systems of the present invention comprises a central
processing unit (CPU), input means, output means, and data storage
means. A skilled artisan can readily appreciate that any one of the
currently available computer-based system are suitable for use in
the present invention. The data storage means may comprise any
manufacture comprising a recording of the present information as
described above, or a memory access means that can access such a
manufacture.
A variety of structural formats for the input and output means can
be used to input and output the information in the computer-based
systems of the present invention. One format for an output means
ranks expression profiles possessing varying degrees of similarity
to a reference expression profile. Such presentation provides a
skilled artisan with a ranking of similarities and identifies the
degree of similarity contained in the test expression profile.
Reagents, Systems and Kits
Also provided are reagents, systems and kits thereof for practicing
one or more of the above-described methods. The subject reagents,
systems and kits thereof may vary greatly. Reagents of interest
include reagents specifically designed for use in production of the
above-described expression profiles of phenotype determinative
genes, i.e., a gene expression evaluation element made up of one or
more reagents. The term system refers to a collection of reagents,
however compiled, e.g., by purchasing the collection of reagents
from the same or different sources. The term kit refers to a
collection of reagents provided, e.g., sold, together.
One type of such reagent is an array of probe nucleic acids in
which the phenotype determinative genes of interest are
represented. A variety of different array formats are known in the
art, with a wide variety of different probe structures, substrate
compositions and attachment technologies. Representative array
structures of interest include those described in U.S. Pat. Nos.
5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806;
5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028;
5,800,992; the disclosures of which are herein incorporated by
reference; as well as WO 95/21265; WO 96/31622; WO 97/10365; WO
97/27317; EP 373 203; and EP 785 280.
In certain embodiments, the arrays include probes for at least 1 of
the genes listed in Tables 1 and/or 2. In certain embodiments, the
number of genes that are from Tables 1 and/or 2 that is represented
on the array is at least 5, at least 10, at least 25, at least 50,
at least 75 or more, including all of the genes listed in Tables 1
and/or 2. The subject arrays may include only those genes that are
listed in Tables 1 and/or 2, or they may include additional genes
that are not listed in Tables 1 and/or 2, such as probes for genes
whose expression pattern can be used to evaluate additional
transplant characteristics, including but not limited to: acute
rejection; chronic allograft injury (chronic rejection) in blood;
immunosuppressive drug toxicity or adverse side effects including
drug-induced hypertension; age or body mass index associated genes
that correlate with renal pathology or account for differences in
recipient age-related graft acceptance; immune tolerance markers in
whole blood; genes found in literature surveys with immune
modulatory roles that may play a role in transplant outcomes (see
e.g., Table 3 for a list of representative additional genes); as
well as other array assay function related genes, e.g., for
assessing sample quality (3'- to 5'-bias in probe location),
sampling error in biopsy-based studies, cell surface markers, and
normalizing genes for calibrating hybridization results; and the
like. Where the subject arrays include probes for such additional
genes, in certain embodiments the number % of additional genes that
are represented and are not directly or indirectly related to
transplantation does not exceed about 50%, usually does not exceed
about 25%. In certain embodiments where additional genes are
included, a great majority of genes in the collection are
transplant characterization genes, where by great majority is meant
at least about 75%, usually at least about 80% and sometimes at
least about 85, 90, 95% or higher, including embodiments where 100%
of the genes in the collection are phenotype determinative genes.
Transplant characterization genes are genes whose expression can be
employed to characterize transplant function in some manner, e.g.,
presence of rejection, etc.
Another type of reagent that is specifically tailored for
generating expression profiles of phenotype determinative genes is
a collection of gene specific primers that is designed to
selectively amplify such genes. Gene specific primers and methods
for using the same are described in U.S. Pat. No. 5,994,076, the
disclosure of which is herein incorporated by reference. Of
particular interest are collections of gene specific primers that
have primers for at least 1 of the genes listed in one Tables 1
and/or 2, often a plurality of these genes, e.g., at least 2, 5,
10, 15 or more. In certain embodiments, the number of genes that
are from Tables 1 and/or 2 that have primers in the collection is
at least 5, at least 10, at least 25, at least 50, at least 75 or
more, including all of the genes listed in Tables 1 and/or 2. The
subject gene specific primer collections may include only those
genes that are listed in Tables 1 and/or 2, or they may include
primers for additional genes that are not listed in Tables 1 and/or
2, such as probes for genes whose expression pattern can be used to
evaluate additional transplant characteristics, including but not
limited to: acute rejection; chronic allograft injury (chronic
rejection) in blood; immunosuppressive drug toxicity or adverse
side effects including drug-induced hypertension; age or body mass
index associated genes that correlate with renal pathology or
account for differences in recipient age-related graft acceptance;
immune tolerance markers in whole blood; genes found in literature
surveys with immune modulatory roles that may play a role in
transplant outcomes (see e.g., Table 3 for a list of representative
additional genes); as well as other array assay function related
genes, e.g., for assessing sample quality (3'- to 5'-bias in probe
location), sampling error in biopsy-based studies, cell surface
markers, and normalizing genes for calibrating hybridization
results; and the like. Where the subject arrays include probes for
such additional genes, in certain embodiments the number % of
additional genes that are represented and are not directly or
indirectly related to transplantation does not exceed about 50%,
usually does not exceed about 25%. In certain embodiments where
additional genes are included, a great majority of genes in the
collection are transplant characterization genes, where by great
majority is meant at least about 75%, usually at least about 80%
and sometimes at least about 85, 90, 95% or higher, including
embodiments where 100% of the genes in the collection are phenotype
determinative genes.
The systems and kits of the subject invention may include the
above-described arrays and/or gene specific primer collections. The
systems and kits may further include one or more additional
reagents employed in the various methods, such as primers for
generating target nucleic acids, dNTPs and/or rNTPs, which may be
either premixed or separate, one or more uniquely labeled dNTPs
and/or rNTPs, such as biotinylated or Cy3 or Cy5 tagged dNTPs, gold
or silver particles with different scattering spectra, or other
post synthesis labeling reagent, such as chemically active
derivatives of fluorescent dyes, enzymes, such as reverse
transcriptases, DNA polymerases, RNA polymerases, and the like,
various buffer mediums, e.g. hybridization and washing buffers,
prefabricated probe arrays, labeled probe purification reagents and
components, like spin columns, etc., signal generation and
detection reagents, e.g. streptavidin-alkaline phosphatase
conjugate, chemifluorescent or chemiluminescent substrate, and the
like.
The subject systems and kits may also include a phenotype
determination element, which element is, in many embodiments, a
reference or control expression profile that can be employed, e.g.,
by a suitable computing means, to make a phenotype determination
based on an "input" expression profile, e.g., that has been
determined with the above described gene expression evaluation
element. Representative phenotype determination elements include
databases of expression profiles, e.g., reference or control
profiles, as described above.
In addition to the above components, the subject kits will further
include instructions for practicing the subject methods. These
instructions may be present in the subject kits in a variety of
forms, one or more of which may be present in the kit. One form in
which these instructions may be present is as printed information
on a suitable medium or substrate, e.g., a piece or pieces of paper
on which the information is printed, in the packaging of the kit,
in a package insert, etc. Yet another means would be a computer
readable medium, e.g., diskette, CD, etc., on which the information
has been recorded. Yet another means that may be present is a
website address which may be used via the internet to access the
information at a removed site. Any convenient means may be present
in the kits.
The following examples are offered by way of illustration and not
by way of limitation.
EXPERIMENTAL
I. Introduction
The objective of this study was to determine whether gene
expression markers could be identified in RNA extracted from
peripheral blood leukocytes (PBL) or renal biopsies predictive of
future graft loss following AR.
II. Array Experiments
Each microarray contained approximately 32,000 DNA spots
representing approximately 12,440 human genes. Total RNA was
isolated (Tri Reagent; MRC Inc., Cincinnati, Ohio) from buffy coats
isolated from whole blood samples. A common reference RNA pool
(Perou et al., Nature (2000) 406:747-52) was used as an internal
standard. Sample or reference RNA were subjected to two successive
rounds of amplification before hybridization to microarrays using
an improved protocol based on the method described by Wang et al
(please provide entire cite). Array data for 62 renal biopsy
samples and 56 whole blood samples were stored in the Stanford
Microarray database (Sherlock et al., Nuc. Acids Res. (2001)
29:152-55) and gene lists filtered at retrieval to provide
expression markers with high fidelity. The two groups of samples
were analyzed in two separate studies. All PBL were used for
initial unsupervised hierarchical clustering (Eisen et al., Proc.
Nat'l Acad. Sci. USA (1998) 95:14863-8), for subsequent supervised
analyses between groups (Significance Analysis of Microarrays; SAM
(Tusher et al., Proc. Nat'l Acad. Sci. USA (2001) 98:5116-21).
III. Customizing a Minimal Gene-Set for AR Class Prediction and
Risk Assessment
We used Predictive Analysis of Microarrays (PAM) (Tusher et al.,
supra) to identify only 97 genes within the renal biopsy dataset,
all having >5-fold difference in expression level, which
classify our learning set of 26 AR samples with 100% concordance to
assigned phenotype. Another analysis using a larger set of 3,170
differentially expressed genes identifies the 33 classifiers with
similar power (FIGS. 1A and 1B). Reproducibility of the diagnostic
signature, in particular within the majority of the AR-1 samples,
is evident by the short branches in the cluster dendogram. AR
expression overlaps with the innate immune response to infection,
as evidenced by cluster analysis and by differential expression of
several TGF-.beta.-modulated genes including RANTES, MIC-1, several
cytokines, chemokines, and cell-adhesion molecules. AR-1 is the
most severe class with the highest rate of graft loss and highest
expression of B-cell specific genes. AR-2 resembles a drug-toxicity
signature and also co-clusters with patients with active viral
infections. The most striking feature of AR-3 is the expression of
genes involved in cellular proliferation and cell cycling
suggesting active tissue repair and regeneration. The presence of
proliferating-cell nuclear antigen (PCNA), a marker of cell
proliferation, was confirmed in all AR-3 samples tested (Sarwal et
al. New Engl. J. Med. 2003 349(2):125-38).
The PAM classification scores grouped the samples with 100%
concordance to assigned classes and reported scores are aligned
with the clustered samples (FIG. 1B). In addition, all 33 genes
selected by PAM have Significance Analysis of Microarrays
significance scores of 0.09% or lower suggesting that they would be
highly significant biomarkers for a customized array list.
A. PAM Class Prediction--
PAM class prediction has also proven to be a powerful approach to
identify putative biomarkers for graft recovery and graft loss. We
have used both Cox-regression and PAM to correlate expression
differences with graft outcome with good success. Both methods
yield significant results in Kaplan-Meier survival analysis
although at the initial 2-year follow-up genes identified by PAM
also yield greater significance. (FIG. 2--Kaplan-Meier survival
analysis for graft loss (red) and no-loss (blue. The genes include
ICAM5-FIG. 2A; (p=0.007), IL6R; FIG. 2B; (p=0.003), STAT1; FIG. 2C;
(p=0.036), and STAT6; FIG. 2D; (p=0.020)).
The gene signature is dominated by increased expression of cell
adhesion genes, selected cytokines, B-cell genes, representatives
in the STAT signaling pathway and several immune response genes
including multiple representatives of both class I and class II HLA
genes.
Representative genes include those from HLA class I (HLA-F, HLA-G),
HLA class II (HLA-DRB1, HLA-DRB5, HLA-DRB4), signal transducers
(STAT1, STAT6), immunoglobulin genes (IGKC, IGHG3), and 2
interferon gamma induced genes (ICAM5, IL6R).
A similar approach was used to identify graft-loss markers in whole
blood samples. The list of the most highly-predictive significant
genes in blood is summarized in Table 4, including the Kaplan-Meier
survival significance score.
TABLE-US-00004 TABLE 4 Fold Unigene Loss/ Symbol Gene ID No-loss
p-value HIST1H2BC Histone 1, H2bc Hs.356901 -3.46 0.00018 IGHG3 Ig
heavy constant gamma 3 (G3m marker) Hs.413826 4.14 0.00134 AHSA2
Activator of heat shock ATPase Hs.122440 2.91 0.00041 TNFRSF10D TNF
receptor superfamily 10b Hs.129844 -2.55 0.00010 MAPK9
Mitogen-activated protein kinase 9 Hs.348446 8.14 0.00444 IFNAR2
Interferon (alpha, beta and omega) receptor 2 Hs.86958 -2.37
0.01760 TM4SF9 Transmembrane 4 superfamily member 9 Hs.8037 -15.29
0.00580 MIF Macrophage migration inhibitory factor Hs.407995 -2.31
0.00674 SCYE1 Small inducible cytokine (Monocyte-activating)
Hs.105656 2.51 0.00154 MAPK1 Mitogen-activated protein kinase 1
Hs.324473 -2.32 0.00019 TGFBR3 TGFb receptor III (betaglycan)
Hs.342874 -2.94 0.00318 IGKC Immunoglobulin kappa constant
Hs.377975 2.35 0.00290 IL1R2 Interleukin 1 receptor, type II
Hs.25333 -4.06 0.01762 IGL Immunoglobulin lambda light chain 3.04
0.02093
The Kaplan-Meier survival curves for 8 of these genes are
illustrated in FIG. 3. The genes in FIG. 3 include A) AHSA2, B)
IGHG1, C) IFNAR2, D) IGKC, E) HIST1H2BC, F) IL1R2, G) MAPK1, and H)
MAPK9.
The functional composition of genes associated with acute
rejection, predicted by analysis of Gene Ontology annotations, is
summarized in Table 5.
TABLE-US-00005 TABLE 5 Genes on EASE Fisher Gene Category Genes
Array Score Exact defense response 105 747 7.15E-12 3.35E-12
response to stimulus/ 152 1482 0.00000108 7.24E-07 acute phase
response apoptosis 50 361 0.00000772 3.63E-06 cell cycle 71 597
0.0000174 9.84E-06 cell proliferation 96 899 0.0000403 0.0000256
protein metabolism 176 1941 0.000228 0.000172 antigen presentation
9 29 0.000707 0.000123 cell growth and/or 244 2887 0.000766
0.000623 maintenance phosphorylation 53 512 0.00539 0.00353 protein
modification 84 902 0.00775 0.00545 hemopoiesis 10 53 0.0116
0.00374 DNA replication 17 122 0.0125 0.00571 B-cell activation 6
22 0.0171 0.00356
The full list of known genes (in ranked order) in whole blood that
are predictive of graft loss following acute rejection is
summarized in Table 1. Of the 81 cDNA clones identified to have the
highest predictive power, 62 are of known function or assigned
unique Unigene Cluster IDs. Similarly, the list of known genes
identified in renal biopsies predictive of graft loss following
acute rejection is summarized in Table 2 (including 30 unique genes
of known function from the 50 cDNA associated clones).
IV. Generation of a Transplant Custom Expression Chip
TxChip
We have compiled the gene lists described in this document for AR
and graft loss along with other expression-based markers identified
to be associated with clinical outcomes and severity of:
1. Acute rejection--including markers associated with graft loss
and/or rate of recovery of renal function following AR (Table
3);
2. Chronic allograft injury (chronic rejection) in blood (Table
3);
3. Immunosuppressive drug toxicity or adverse side effects
including drug-induced hypertension (Table 3);
4. Age or body mass index associated genes that correlate with
renal pathology or account for differences in recipient age-related
graft acceptance (Table 3);
5. Immune tolerance markers in whole blood (Table 3);
6. Control genes for assessing sample quality (3'- to 5'-bias in
probe location), sampling error in biopsy-based studies, cell
surface markers, and normalizing genes for calibrating
hybridization results;
7. Genes found in literature surveys with immune modulatory roles
that may play a role in transplant outcomes (Table 3) to produce
the list for a representative array having probes to genes listed
in Table 3.
A. Test of Expression Uniformity Across a Pilot Study of Renal
Biopsies.
In the identification of the gene markers described in this
invention disclosure, we compared the expression across a set of 67
renal biopsies described in detail by our laboratory. A subset of
the biopsy-generated gene expression markers was used clustered to
compare expression profiles in patients with confirmed cases of DT,
CAN, AR and no significant abnormality (Normal). These patients
were on two very different immunosuppressant regimes, either
steroid-based or steroid-free (clinical regiment previously
described in (Sarwal et al., Transplantation (2001) 72:13-21) and
Sarwal et al., Transplantation (2003) 76:1331-9).
FIG. 4 illustrates that the gene expression is generally
uniform/consistent across the full clinical groups analyzed as the
gene expression levels segregate well within patient groups.
Further, within each group (DT, CAN, AR or Normal) expression
levels of these marker genes are independent of immunosuppression
use.
The 479 gene list of Table 3 comprises design and specification for
a customized thematic Transplant Chip (TxChip V1) and full-length
mRNA sequences for these genes are listed in Table 3. The gene
listing is cross-indexed to the studies listed above. We observe a
modest overlap in the list of informative genes. For example,
expression levels of IGHM positively correlate with acute rejection
risk and negatively correlate with immune tolerance. An advantage
of having the full compilation of genes on a common platform is
that new discoveries like this can be made in future studies.
It is evident that subject invention provides a convenient and
effective way of determining whether a graft in a subject will
survive, e.g., following acute rejection. As such, the subject
invention provides a number of distinct benefits, including the
ability to identify clinically relevant AR groups with differing
therapeutic responses and prognosis, and allow for individualized
treatment and monitoring. As such, the subject invention represents
a significant contribution to the art.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it is readily apparent to those of ordinary skill in
the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
* * * * *
References