Noninvasive Prediction of
Acute Rejection of Renal Allografts
M. Suthanthiran, M.D.
Stanton Griffis Distinguished Professor
Chief, Nephrology and Hypertension
Chief, Transplantation Medicine
New York Presbyterian - Cornell
What Does a Transplant Clinician Want?
(Besides an Outstanding Renal Pathologist)
• Repetitively ascertain allograft status by noninvasive means
• Anticipate rejection
• Prognosticate rejection
• Mechanism-based therapy
• Predict allograft function
• Immunosuppression weaning or reintroduction of therapy
• Individualize/Optimize immunosuppression
Invention of The Polymerase Chain Reaction
1993 Nobel Prize in Chemistry
Kary B. Mullis
Development of Competitive Quantitative PCR Assay
Li et al. J Exp Med 1991
Development of Urinary Cell mRNA Profiling Protocol
( Li et al. N Engl J Med 2001)
50 cc urine specimen
Urine sediment, isolation of
total RNA
Reverse transcription to
cDNA
Urinary cell levels of mRNA
measured using competitive
quantitative PCR assays
Urinary Cell CTL mRNA Profiling Study
•
Urine Specimens and Renal Allograft Recipients : 151 Specimens from
85 Recipients.
24 specimens from 22 recipients with biopsy confirmed acute rejection; 5 from
5 recipients with CAN; 15 from 11 recipients with Other findings; and 107
specimens from 47 recipients with stable allograft function.
•
Design and Development of Competitive Quantitative PCR Assays:
Levels of mRNA for granzyme B, perforin and cyclophylin B in urinary cells
were measured using gene specific primer pairs and gene specific competitor
DNA construct.
•
Statistical Analysis: Transcript levels were expressed in femtogram of
mRNA per microgram of total RNA, and log transformed to reduce skew. Oneway mixed – level ANOVA and Dunnett’s test for multiple comparisons were
used. Receiver-operating-characteristic curve analysis was used to determine
cutoff points yielding the highest combined sensitivity and specificity for
predicting an episode of acute rejection.
HYPOTHESIS
Measurement of mRNA encoding cytotoxic
proteins granzyme B and perforin offers a
noninvasive means of diagnosing acute
rejection of renal allografts.
Levels of mRNA in Urinary cells
Box and whisker plots show the
10th, 25th, 50th (median) and 90th
percentile values for perforin
mRNA, granzyme B mRNA and
cyclophilin B mRNA in urine
samples from patients classified
as having acute rejection, Other
findings, chronic allograft
nephropathy or a stable course
after transplantation.
The levels of perforin and
granzyme B, but not those of
cyclophilin B, were significantly
higher in the patients with an
episode of acute rejection than
in the other groups (P=0.001, by
one-way mixed-level analysis of
variance).
Values in parenthesis are the
numbers of urine samples. In all
cases log-transformed values
are shown
Li et al. N Engl J Med 2001
Receiver-Operating-Characteristic Curves for mRNA Levels
The fraction of true positive
results (sensitivity) and false
positive results (1 – specificity)
for perforin mRNA levels,
granzyme B mRNA levels, and
cyclophilin B mRNA levels as
markers of acute rejection are
shown.
Acute Rejection could be
predicted with a sensitivity of
83% and a specificity of 83%
with the use of a cutoff value of
0.9 fg of perforin mRNA per
microgram of total RNA , and
with a sensitivity of 79% and a
specificity of 77% with the use
of a cutoff value of 0.4 fg of
granzyme B mRNA per
microgram of total RNA.
Li et al. N Engl J Med 2001
Levels of mRNA in Sequential Urine Samples
Perforin mRNA, granzyme B mRNA, and
cyclophilin B mRNA were measured in
urine samples obtained during the first nine
days after transplantation.
The mean (±SE) levels of perforin mRNA
and granzyme B mRNA but not those of
cyclophilin B mRNA were higher in the 8
patients in whom acute rejection
developed within the first 10 days after
transplantation than in the 29 patients in
whom acute rejection did not develop
within the first 10 days after
transplantation.
The respective numbers of urine samples
obtained from the patients with an episode
of acute rejection and those without such
an episode were as follows: 6 and 43 on
day 1, 2, or 3 after transplantation; 5 and
26 on day 4, 5, or 6; and 6 and 14 on day
7, 8, or 9.
Means, standard errors, and P values were
estimated with use of a mixed-level twoway analysis of variance. In all cases, logtransformed values are shown.
Li et al. N Engl J Med 2001
Summary of Findings
•
Noninvasive diagnosis of acute rejection is feasible by measurement of mRNA
for perforin and granzyme B in urine.
•
Measurement of mRNAs in sequential urine specimens may predict the
development of acute rejection.
•
No significant association exists between mRNA levels and biopsy grade or the
time from kidney transplantation to the development of acute rejection time.
•
Graft dysfunction due to non-immulogical causes (e.g., ATN) is not associated
with an increase in urinary cell levels of perforin or granzyme B mRNA.
•
Noninvasive diagnosis of BKV nephropathy is feasible by measurement of
mRNA for BKV VP1 mRNA in urine (Ding et al. Transplantation 2002).
•
Bacterial urinary tract infection is not associated with an increase in urinary
cell levels of perforin and granzyme B (Dadhania et al. Transplantation 2003).
Allograft Directed Immune Response
•
Cellular Traffic
(Tatapudi et al. Kidney Intl 2004)
•
Physical Contact
(Ding et al. Transplantation 2003)
•
Target Cell Damage
(Muthukumar et al. Transplantation 2003)
•
Protective/Regulatory
Response
FOXP3
• A Specification and functional factor for CD25+CD4+
regulatory T cells (Tregs).
(Fontenot et al. Nat Immunol 2003; Sakaguchi S. Nat Immunol 2005)
• Mutations in human FOXP3 gene result in an
autoimmune disease characterized by
polyendocrinopathy and enteropathy.
(Wildin et al. Nat Genet 2001; Bennett et al. Nat Genet 2001)
• Tregs implicated in experimental transplantation
tolerance.
(Graca et al. J Exp Med 2002; Lee I et al. J Exp Med 2005)
Urinary Cell FOXP3 mRNA Profiling Study
•
Urine Specimens and Renal Allograft Recipients: 83 urine specimens
from 83 renal allograft recipients
36 specimens from 36 recipients with biopsy confirmed acute rejection; 18
from 18 recipients with CAN; and 29 from 29 recipients with stable allograft
function.
•
Development of Pre-amplification Enhanced Real Time Quantitative
PCR Assays: Levels of mRNA for FOXP3, CD25,CD3ε, perforin and 18S
rRNA in urinary cells were quantified using the standard curve method
developed by Ding et al (Transplantation 2002).
•
Statistical Analysis: Transcript levels were log transformed and mRNA
copy numbers normalized with the use of 18S rRNA copy number. KruskalWallis, Dunn’s test for multiple comparisons and Mann-Whitney test were
used for group comparisons. Chi-square test for linear trend in graft failure
rates.
HYPOTHESES
Acute rejection is associated with a lower level of
urinary cell FOXP3 mRNA levels compared to stable
patients
Urinary cell FOXP3 mRNA predict acute rejection
outcome , and identify patients at high risk for graft
loss after an episode of acute rejection.
Levels of FOXP3 mRNA in Urinary Cells
(A)
(B)
12
P<0.001
8
Log CD25 mRNA
Log FOXP3 mRNA
10
6
4
2
0
8
6
4
2
0
-2
-2
Acute
Rejection
(N=36)
CAN
(N=18)
Acute
Rejection
(N=36)
Stable
Graft Function
(N=29)
CAN
(N=18)
Stable
Graft Function
(N=29)
The levels of FOXP3 mRNA, CD25
mRNA, CD3RNA and perforin mRNA
were higher in the urinary cells from
patients with acute rejection than in the
patients with CAN or normal biopsies.
(D)
(C)
12
12
P=<0.001
Log Perforin mRNA
10
Log CD3 mRNA
P<0.001
10
8
6
4
2
0
Box plots show the 10th, 25th, 50th
(median), 75th, and 90th percentile
values for log-transformed FOXP3
mRNA, CD25 mRNA, CD3RNA and
perforin B mRNA in urine samples
obtained from 36 patients with biopsyconfirmed acute rejection, 18 patients
with biopsy-confirmed chronic allograft
nephropathy (CAN) and 29 patients
with stable graft function and normal
allograft biopsies (normal biopsy).
P<0.001
10
8
P-values are based on the KruskalWallis test, treating the logtransformed mRNA levels as the
dependent variable.
6
4
2
0
-2
-2
Acute
Rejection
(N=36)
CAN
(N=18)
Stable
Graft Function
(N=29)
Acute
Rejection
(N=36)
CAN
(N=18)
An inverse relationship was found between FOXP3 mRNA levels and
serum creatinine in the acute rejection group (r=-0.38, P=0.02)
Stable
Graft Function
(N=29)
In all cases log-transformed levels,
normalized by 18S rRNA, are shown.
Muthukumar et al. N Engl J Med 2005
Levels of FOXP3 mRNA in Urinary Cells Predict
Reversal of an Episode of Acute Rejection
(A)
(B)
12
P=0.001
Log CD25 mRNA
Log FOXP3 mRNA
10
6
2
-2
8
4
0
Reversible
Reversible
Non-reversible
Non-reversible
Acute Rejection Episode
Acute Rejection Episode
(N=26)
(N=10)
(N=10)
(N=26)
(C)
(D)
14
14
P=0.35
Log Perforin mRNA
Log CD3 mRNA
Levels of FOXP3 mRNA in urinary cells but
not the levels of CD25 mRNA, CD3 mRNA
and perforin mRNA were significantly
higher in the group with successful
reversal than in the group without reversal.
P=0.22
10
6
2
P=0.43
Non-reversible
Acute Rejection Episode
(N=26)
(N=10)
In logistic – regression analyses predicting
non-response, levels of FOXP3 mRNA in
urinary cells remained significant after
statistical control for serum creatinine level
(P=0.04) and the time from transplantation
to rejection (P=0.02).
A linear combination of FOXP3 and
creatinine was a better predictor of
rejection reversal (90% sensitivity and 96%
specificity) than FOXP3 mRNA levels alone
or serum creatinine levels alone (85%
sensitivity and 90% specificity).
10
6
2
Reversible
FOXP3 mRNA level predicted rejection
reversal with a sensitivity of 90% and a
specificity of 73% (P=0.001). The levels of
mRNA for CD25, CD3 and perforin were not
predictive of reversal of acute rejection.
Reversible
Non-reversible
Acute Rejection Episode
(N=26)
(N=10)
Successful reversal of acute rejection was
not predicted by the subject’s age, sex,
race, graft donor source, Banff grade, or
initial anti-rejection treatment.
Muthukumar et al. N Engl J Med 2005
Relative Risk of Graft Failure after an Episode of Acute Rejection
The rates and relative risks of
graft failure within six months
after an episode of acute
rejection for thirds of FOXP3,
CD25, CD3 , and perforin
mRNA levels are shown.
The graft-failure rate was 50
percent and the relative risk
was 6 for the lowest third of
FOXP3 mRNA levels; 25
percent and 3, respectively, for
the middle third; and 8 percent
for the highest third (P=0.02 by
the chi-square test for linear
trend) (Panel A).
There was no relationship
between graft failure after an
episode of acute rejection and
the thirds of mRNA levels for
CD25, CD3 , and perforin
(Panels B, C, and D).
Muthukumar et al. N Engl J Med 2005
Summary of Findings
•
The levels of FOXP3 mRNA are higher in the urinary cells from patients with
acute rejection than in the patients with CAN or normal biopsies.
•
A significant inverse relationship exists between FOXP3 mRNA levels and
serum creatinine measured during an episode of in the acute rejection.
•
Measurement of FOXP3 mRNA in urine offers a noninvasive means of
improving the prediction of outcome of acute rejection of renal transplants.
•
A linear combination of FOXP3 and creatinine is a better predictor of rejection
reversal (90% sensitivity and 96% specificity) than FOXP3 mRNA levels alone
(90% sensitivity and 73% specificity) or serum creatinine levels alone (85%
sensitivity and 90% specificity).
•
A strong inverse relationship exists between levels of FOXP3 mRNA in urinary
cells and the time from kidney transplantation to the development of acute
rejection (r=-0.42, P=0.01).
Tolerance Signature
Intragraft levels of FOXP3 mRNA in Human Renal Allografts
Total RNA was isolated from renal-allograft biopsy specimens and
reverse transcribed to cDNA, and levels of mRNA were measured
with the use of pre-amplification enhanced real-time quantitative
polymerase-chain-reaction assays.
A total of 23 biopsy specimens were examined for intragraft levels
of FOXP3 mRNA, granzyme B mRNA, and housekeeping gene
18S ribosomal RNA (18S rRNA). Of the 23 biopsy specimens, 6
were obtained from four patients with stable renal-allograft function
who were not receiving immunosuppressive therapy (IS) (stable ISfree group). Eight biopsy specimens were obtained from eight
patients with stable renal-allograft function and normal protocol
biopsy results; these patients were receiving maintenance
immunosuppressive drug therapy comprising tacrolimus and
mycophenolate mofetil (stable-with-IS group). Five biopsy
specimens were obtained from five kidney donors (normal-kidney
group), and four biopsy specimens were obtained from four
patients with biopsy-confirmed acute rejection (acute-rejection
group).
The mRNA copies were log transformed and normalized with 18S
rRNA. Data analysis using Nested ANOVA and Tukey’s HSD
showed that the intragraft level of FOXP3 mRNA in the Stable ISfree group was higher compared to the Stable with IS group
(P=0.007), or the Normal Kidney group (P=0.0002), but lower than
in the Acute Rejection group (P<0.0001). Intragraft level of
granzyme B mRNA in the Stable IS-free group was not different
from that in the Stable with IS group (P=0.91), or the Normal
Kidney group (P=0.36), and lower than in the Acute Rejection
group (P=0.0002).
Kawai et al. N Engl J Med 2008
Allograft Status: A Time-line Model
Time - Lines for Diagnosis
Molecular
Histological
Clinical
Time - Lines for Treatment Efficacy ?
mRNA Expression Signatures
Predictive of Allograft Status
“Ceterum censeo Carthaginem esse delendam”
•
Diagnose rejection by noninvasive means
•
Anticipate rejection
•
Identification of rejection prior to tissue injury
•
Mechanism-based therapy
•
Prognosticate rejection
•
Predict Allograft function
•
Informed weaning or reintroduction of therapy
•
Individualize/Optimize immunosuppression