Transplantation I
Physiologic, Histologic, and Pharmacologic Markers of Graft Function
Background
Graft
survival for all solid organ transplantation procedures is restricted by
acute and chronic rejections. The
solution to this problem is induction of a state of donor-specific
tolerance in the patient so rejections will not occur.
Current methods of diagnosing allograft dysfunction are inadequate
in that significant organ damage occurs prior to the establishment of a
clinical diagnosis. Clinical
tolerance remains an elusive goal despite success in animal models.
One of the main hurdles in developing tolerance strategies is the
lack of a clinical biomarker or a "tolerance assay." The development of assays or novel technologies that will
enable detection of allograft dysfunction/rejection, monitor responses to
therapy, and predict long-term
Objectives
• Assess graft dysfunction for renal, hepatic,
and cardiac allografts by histological criteria and identify newer methods
to quantitatively assess the degree of dysfunction
•
Define physiological and pharmacological criteria for graft
dysfunction and validate the techniques
•
Identify areas in need of further diagnostic tool refinement
Agenda
Moderators:
Amir Tejani, M.D., New York Medical College
John Neylan, M.D., Emory University Hospital
Introduction
Stephen
M. Rose, Ph.D., National Institute of Allergy and Infectious Diseases
Physiologic, Histologic, and Pharmacologic Markers of Graft Function
M.
Roy First, M.D., University of Cincinnati Medical Center
Markers of Hepatic Function/Rejection
John
R. Lake, M.D., University of Minnesota
Use of Surrogate Endpoints in Cardiac Transplantation
Leslie
W. Miller, M.D., University of Minnesota
Break
Pharmacokinetic and Pharmacodynamic Surrogates in Transplantation
Barry D. Kahan, Ph.D., M.D., University of Texas Medical School,
Houston
Optimal Pharmacological Monitoring of Antirejection DrugsGiuseppe
Remuzzi, M.D., Negri Bergamo Laboratories, Italy
Prediction of Long-Term Renal Allograft Outcome Using Image Analysis of
Sirius Red Staining in Protocol Biopsies
Paul
C. Grimm, M.D., University of California, San Diego
Immune Parameters Correlating Hyporesponsiveness
Ronald
H. Kerman, Ph.D., University of Texas Medical School
Open
Discussion
Summary of Session Recommendations
AGENDA
Physiologic,
Histologic, and Pharmacologic Markers of Graft Function
M.
Roy First, M.D
Kidney
transplantation outcomes have improved progressively over the past three
decades. However, despite a
large body of literature produced during this period, few reports indicate
agreement on the clinical presentation of acute rejection, the treatment
of acute rejection, the response of acute rejection to therapy, and the
correlations between the pathologic findings and the clinical presentation
and response to treatment. In
the precyclosporine era, a number of clinical parameters associated with a
diagnosis of acute rejection were described.
However, after the introduction of cyclosporine, these became less
obvious in the patient experiencing an acute rejection episode.
Measurement of serum creatinine has been the most significant
biochemical marker of acute rejection, but considerable tissue damage may
occur prior to the serum creatinine becoming elevated.
A search for a more reliable biochemical marker of graft
dysfunction remains elusive, and histologic assessment of the allograft
has therefore become the gold standard for the diagnosis of acute
rejection. The Banff
classification of acute rejection grades the process according to
histologic severity. A strong
correlation has been shown between the histologic severity and clinical
and biochemical parameters and provides a reliable means for stratifying
patient risk of treatment success or failure.
By using the pathologic findings in conjunction with other markers
of acute rejection, the clinician should be able to make the decision on
treatment so as to offer the patient the maximum benefit of judicious
antirejection therapy, while avoiding unnecessary overimmunosuppression in
the absence of data supporting the benefit of such therapy to an
individual patient.
Key
References
Al‑Awwa
IA, Hariharan S, First MR. Importance of allograft biopsy in renal
transplant recipients: Correlation between clinical and histologic
diagnosis. Am J Kidney Dis 1998;31(suppl 1):S15‑S18.
Gaber
LW, Moore LW, Gaber AO, First MR, Guttmann RD, Pouletty P, Schroeder TJ,
Soulillou JP. Utility of standardized histological classification in the
management of acute rejection. 1995 Efficacy Endpoints Conference.
Transplantation 1998;64:376‑380.
Guttmann
RD, Soulillou JP. Definitions of acute rejection and controlled clinical
trials in the medical literature. Am J Kidney Dis 1998;31(suppl
1):S3‑S6.
Guttman
RD, Soulillou JP, Moore LW, First MR, Gaber AO, Pouletty P, Schroeder TJ.
Proposed consensus for definitions and endpoints for clinical trials of
acute kidney transplant rejection. Am J Kidney Dis 1998;31(suppl
1):S40‑S46.
Solez K, Axelsen RA, Benediktsson H, Burdick JF, Cohen AH, Colvin RB, Croker BP, Droz D, Dunnill MS, Halloran PF, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int 1993;44:411‑422.
Use
of Surrogate Endpoints in Cardiac Transplantation Abstract
Leslie
W. Miller, M.D.
Acute
cellular rejection and chronic allograft rejection (allograft coronary
disease) are the two primary endpoints in most trials in heart
transplantation. Unlike renal
transplantation, there this no biochemical marker to suggest or define
rejection, and therefore, endomyocardial biopsy has remained the gold
standard for diagnosis of rejection in heart transplantation for the past
25 years. However, there is limited evidence to suggest that the
incidence, frequency, severity, or time to rejection have a good
correlation with graft function, survival, or development of chronic
rejection. Perhaps the most
definitive study regarding the correlation between acute and chronic
rejection was with the use of intravascular ultrasound to measure direct
intimal thickening within the allograft vessel as the marker of chronic
rejection. Only the average biopsy score in the first 3 months
posttransplant had any correlation with development of intimal thickening
using the current grading system to define acute cellular rejection.
Function of the graft (ejection fraction) can be readily measured,
but patients who demonstrate evidence of severe graft dysfunction or
hemodynamic compromise have a 40 to 50 percent mortality at 1 year.
Therefore, all heart transplant recipients are biopsied on a
predetermined protocol basis in hopes of detecting rejection before graft
dysfunction develops. Data
from protocol kidney biopsies, which were not driven by change in
function, have confirmed a significant “trafficking” of lymphocytes
and inflammatory cells that when left untreated often resolve entirely and
were never associated with the change in function.
These data suggest that many of the biopsies interpreted as showing
histologic rejection in heart transplantation may represent a potentially
innocent immunological response. This
phenomenon is one of the main reasons for the lack of correlation of acute
rejection with chronic rejection and the problem with the use of
biopsy‑proven rejection as a primary endpoint in heart transplant
trials. A number of
noninvasive or surrogate endpoints have been examined in heart
transplantation, including
(1) echocardiography to define alterations in dystocic compliance
that may precede overt systolic dysfunction; (2) measurement of voltage
from endocardial electrodes; and (3) markers of immune activation,
including originally simple T‑cell subsets, but more recently both
surface and soluble interleukin‑2 receptor.
Other approaches have included examination of proinflammatory
cytokines, such as tumor necrosis factor and IL‑6, as well as
adhesion molecules ICAM and VCAM. The
most important advance in use of biologic markers as endpoints for
defining chronic allograft rejection in heart transplant recipients is the
use of intervascular ultrasound. This
new technology allows direct examination and measurement of the amount of
intimal thickening within the allograft vessels.
This safe and highly reproducible technology has now become the
most definitive surrogate for chronic rejection in any vascularized
allograft. The major deficiency in the field of transplantation is the
lack of a bioassay of the level of immunosuppression. One recent approach is the use of a cell line expressing
donor antigens to which a mixed lymphocyte culture can be performed at any
time following transplantation to assess the degree of donor specific
alloreactivity. This test not
only provides a relative quantitation of low, medium, or high reactivity
but also can be used as a target for increasing or decreasing
immunosuppression.
Key
References
Addonizio
L. Detection of cardiac allograft rejection using radionuclide techniques.
Prog Cardiovasc Dis 1990;33(2):73‑83.
Carlos
T, Gordon D, Fishbein D, Himes V, Coday A, Ross R, Allen M. Vascular cell
adhesion molecule‑1 is induced on endothelium during acute rejection
in human cardiac allografts. J Heart Lung Transplant
1992;11:1103‑1109.
Costanzo‑Nordin
MR. Cardiac allograft vasculopathy: Relationship with acute cellular
rejection and histocompatibility. J Heart Lung Transplant
1992;11(suppl):S90‑103.
Griffiths
G, Namikawa R, Mueller C, Liu C, Young J, Billingham M, Weissman I.
Granzyme A and perforin as markers for rejection in cardiac
transplantation. Eur J Immunol 1992;21:687‑692.
Hauptman
P, Nakagawa T, Tanaka H, Libby P. Acute rejection: Culprit or coincidence
in the pathogenesis of cardiac graft vascular disease? J Heart Lung
Transplant 1995;14:S173‑S180.
Hosenpud
J. Noninvasive diagnosis of cardiac allograft rejection: Another of many
searches for the grail. Circulation 1992;85(1):368‑371.
Kobashigawa
J, Miller L, Yeung A, Hauptman P, Ventura H, Wilensky R, Valantine H,
Wiedermann J, and the Sandoz/CVIS Investigators. Does acute rejection
correlate with the development of transplant coronary artery disease? A
multicenter study using intravascular ultrasound. J Heart Lung Transplant
1995;14:S221‑226.
Schuetz
A, Fritsch S, Kemkes B, Kugler C, Angermann C, Spes C, Anthuber M, Weiler
A, Wenke K, Gokel J. Antimyosin monoclonal antibodies for early detection
of cardiac allograft rejection. J Heart Transplant 1990;9:654‑661.
Valantine
H, Fowler M, Hunt S, Naasz C, Hatle L, Billingham M, Stinson E, Popp R.
Changes in Doppler echocardiographic indexes of left ventricular function
as potential markers of acute cardiac rejection. Circulation 1987;76(suppl
V):V‑86.
Warnecke
H, Schüler S, Goetze H, Matheis G, Süthoff U, Müller J, Tietze U,
Hetzer R. Noninvasive
monitoring of cardiac allograft rejection by intramyocardial electrogram
recordings. Circulation
1986;74(suppl III):III‑72.
Young
J, Windsor N, Smart F, Kleiman N, Weilbaecher D, Noon G, Nelson D,
Lawrence E. Inability of
isolated soluble interleukin‑2 receptor levels to predict biopsy
rejection scores after heart transplantation. Transplantation
1991;51(3):636‑641.
Pharmacokinetic
and Pharmacodynamic Surrogates in Transplantation
Barry
D. Kahan, Ph.D., M.D.
The
advent of a variety of novel immunosuppressive agents has led to a need to
understand their pharmacokinetics and pharmacodynamics when either used
alone or in drug combinations. Initial
data that the pharmacokinetic behavior of an immunosuppressive drug is
important to predict outcome were first obtained with cyclosporine (CsA) (Kahan
et al. 1982, 1983, 1984). Almost
20 years of investigation have shown that concentration rather than dose
determines outcome: A low
drug exposure represents a risk factor for acute rejection episodes (Lindholm
and Kahan 1993) and a variable exposure, a risk factor for chronic
rejection (Kahan et al. 1996). Parallel
considerations may be important for the dosing of tacrolimus,
mycophenolate mofetil, and possibly sirolimus (Napoli and Kahan 1996).
Pharmacodynamic assays to quantitate drug effects on transplant
recipient lymphoid cells have been limited due to the rapid reversibility
of the effects, general insensitivity of the assays, and the difficulty to
assay cells ex vivo without altering their pathophysiologic state.
The largest effort has been reported with CsA estimating IL‑2
m‑RNA content by Southern blots with specific probes (Yoshimura et
al. 1987), measuring cytokine production by patient lymphocytes (Yoshimura
and Kahan 1985), and most recently by in vitro calcineurin assays (Batiuk
et al. 1995). Using the
median effect equation to obtain a rigorous model of drug action, one can
evaluate the immunosuppressive as well as toxic interactions between two
agents as more than additive (synergistic), additive, or less than
additive (antagonistic) (Kahan 1985).
Due to the possibility of interactions of CsA and sirolimus at the
tissue level—namely, pharmacokinetic interactions at cytochrome P450 3A4
(Stepkowski et al. 1996) or pharmacodynamic interactions at the level of
low‑density lipoprotein generation and metabolism—we have recently
developed new mathematical models that describe combined pharmacokinetic
and pharmacodynamic effects.
Key
References
Batiuk
TD, Pazderka F, Enns J, Decastro L, Halloran PF. Cyclosporine inhibition
of calcineurin activity in human leukocytes in vivo is rapidly reversible.
J Clin Invest 1995;96:1254‑1260.
Kahan
BD. Immunologic monitoring: Utility and limitations. Transplant Proc
1985;17:1537‑1545.
Kahan
BD, Ried M, Newburger J. Pharmacokinetics of cyclosporine in human renal
transplantation. Transplant Proc 1983;15:446‑453.
Kahan
BD, Van Buren CT, Lin SN, Ono Y, Agostino G, LeGrue SJ, Boileau M, Payne
WD, Kerman RH. Immunopharmacological monitoring of cyclosporin
A‑treated recipients of cadaveric kidney allografts. Transplantation
1982;34:36‑45.
Kahan
BD, Welsh M, Schoenberg L, Rutzky L, Katz SM, Urbauer DL, Van Buren CT.
Variable oral absorption of cyclosporine: A biopharmaceutical risk factor
for chronic renal allograft rejection. Transplantation
1996;62:599‑606.
Kahan
BD, Wideman CA, Ried M, Gibbons S, Jarowenko MV, Flechner SM, Van Buren
CT. The value of serial serum trough cyclosporine levels in human renal
transplantation. Transplant Proc 1984;16:1195‑1199.
Lindholm
A, Kahan BD. Influence of cyclosporine pharmacokinetic parameters, trough
concentrations and AUC monitoring on outcome after kidney transplantation.
Clin Pharmacol Ther 1993;54:205‑218.
Napoli
KL, Kahan BD. Routine clinical monitoring of sirolimus (rapamycin)
whole‑blood concentrations by HPLC with ultraviolet detection. Clin
Chem 1996;42:1943‑1948.
Stepkowski
SM, Napoli KL, Wang ME, Qu X, Chou TC, Kahan BD. Effects of the
pharmacokinetic interaction between orally administered sirolimus and
cyclosporine on the synergistic prolongation of heart allograft survival
in rats. Transplantation 1996;62:986‑994.
Yoshimura
N, Kahan BD. Pharmacodynamic assessment of in vivo cyclosporine effect on
interleukin‑2 production by lymphocytes of kidney transplant
recipients. Transplantation 1985;40:661‑666.
Yoshimura
N, Oka T, Clark SC, Kahan BD. The inhibition of IL‑2 gene expression
at the level of messenger RNA by in vivo cyclosporine treatment in kidney
transplant recipients. Transplant Proc 1987;19:3510‑3512.
Optimal Pharmacological Monitoring of Antirejection
Drugs
Giuseppe
Remuzzi, M.D.
Pharmacological monitoring is a key step
in the management of transplant recipients to allow adequate
immunosuppression to avoid graft rejection and minimize drug toxicity. Therapeutic monitoring of trough blood cyclosporine (CsA)
concentration has been widely adopted to adjust CsA dose in individual
subjects. However,
trough‑level monitoring is not of universal help.
More informative than trough CsA concentration is the area under
the concentration‑time curve (AUC), which is calculated from the
individual complete pharmacokinetic profile.
However, this approach is quite expensive and time consuming and
increases discomfort for the patients, making it seldom feasible in
routine outpatient clinic monitoring.
Thus, abbreviated CsA AUC profiles have been proposed. Recent data show the possibility of accurately estimating the
CsA AUC using only three very early blood samples after Neoral dosing.
Attempts to define abbreviated kinetic profiles in AUC monitoring
has also been extended to the more recent immunosuppressants that are now
entering routine clinical application.
This is the case of tacrolimus, mycophenolate mofetil, as well as
sirolimus, for which a limited sampling strategy represents an efficient
approach to assess total exposure to drugs.
Although the proposed strategies are good predictors, they are
difficult to apply to day‑by‑day drug monitoring in clinical
practice since in all cases the last time‑point of blood sampling is
far from drug dosing (6, 9, or even 12 hours), making the procedure
cumbersome for outpatients and taxing for the transplant centers in terms
of staff effort. Evaluating
drug exposure is the conventional way to optimal pharmacological
monitoring of transplant patients but does not reveal more on the level of
immunosuppression achieved by a given antirejection drug.
Thus, efforts should focus on setting up simple, accurate, and
precise methods for monitoring the level of T‑lymphocyte inhibition
in these circumstances.
Key
References
Amante AJ, Kahan BD. Abbreviated
area‑under‑the‑curve strategy for monitoring
cyclosporine microemulsion therapy in immediate posttransplant period.
Clin Chem 1996;42:1294.
Batiuk TD, Yatscoff RW, Halloran PF. What
is the dose‑response curve for the effects of cyclosporine on
calcineurin and cytokine induction in vivo? Transpl Proc 1994;26:2835.
Fruman D, Klee C, Bierer B. Calcineurin
phosphatase in T lymphocytes is inhibited by FK506 and cyclosporine A.
Proc Natl Acad Sci U S A 1992;89:3686.
Gaspari F, Perico N, Signorini O, Caruso
R, Remuzzi G. Abbreviated kinetic profiles in
area‑under‑the‑curve monitoring of cyclosporine therapy.
Kidney Int 1998;54:2146.
Kaplan B, Meier‑Kriesche H‑U,
Napoli K, Kahan BD. A limited sampling strategy for estimating sirolimus
area‑under‑the‑concentration curve. Clin Chem
1997;43:539.
Keown P, Landsberg D, Halloran P, Shoker
A, Rush D, Jeffery J, et al. A randomized, prospective multicenter,
pharmacoepidemiologic study of cyclosporine microemulsion in stable renal
graft recipients. Report of the Canadian Neoral Transplantation Study
Group. Transplantation 1996;62:1744.
Ku Y‑M, Min DJ. An abbreviated
area‑under‑the‑curve monitoring for tacrolimus in
patients with liver transplants. Ther Drug Monit 1998;20:219.
Lindholm AS, Kahan BD. Influence of
cyclosporine pharmacokinetics, trough concentration, and AUC monitoring on
outcome after kidney transplantation. Clin Pharmacol Ther 1993;54:205.
Perico N, Remuzzi G. Prevention of
transplant rejection. Current treatment guidelines and future
developments. Drugs 1997;54:533.
Schutz E, Armstrong VW, Shipkova M, Weber
L, Niedmann PD, Lammersdorf T, Wiesel M, Mandelbaum A, Zimmerhackl LB,
Mehls O, Tonshoff B, Oellerich M. Limited sampling strategy for the
determination of mycophenolic acid area under the curve in pediatric
kidney recipients. German Study Group on MMF Therapy in Pediatric Renal
Transplant Recipients. Transpl Proc 1998;30:1182.
Prediction of Long‑Term Renal Allograft Outcome
Using Image Analysis of Sirius Red Staining in Protocol Biopsies
Paul
C. Grimm, M.D.
The 6‑month Banff Chronic Score (BCS)
is a predictor of the 24‑month serum creatinine in renal transplant
patients. As components of the Banff Chronic Score are subject to sample
error, computerized image analysis of interstitial fibrosis may allow more
precise quantitation. The
objective of this study was to assess whether quantitation of interstitial
fibrosis by image analysis could predict long‑term graft outcome. We studied 6‑month protocol allograft biopsies from 51
patients with at least 3 years of followup.
1/serum creatinine graphs were used to estimate the time to graft
failure (TTGF) by extrapolating to a creatinine level of 5 mg/dL.
A blinded observer analyzed biopsy fibrosis by using the mean
particle size of Sirius Red‑stained tubulointerstitial collagen
using image analysis with watershed segmentation. The BCS was used as a
comparison. The total BCS of
the 6‑month biopsy was correlated with TTGF (p=0.0011, r=0.44, r2=0.181).
The mean particle size of interstitial Sirius Red staining was also
correlated with TTGF (p=0.0001, r=0.516, r2=0.266).
This study of computerized image analysis indicates a superior
correlation of Sirius Red analysis with TTGF than the BCS.
Further development is necessary to determine whether this will be
useful in predicting allograft outcome.
Key
References
Nickerson P, Jeffery J, Gough J, McKenna
R, Grimm PC, Cheang M, Rush D. Identification of clinical and
istopathological risk factors for diminished renal function 2 years
post‑transplant. J Am Soc Nephrol 1998;9:482‑487.
Rush D, Nickerson P, Gough J, McKenna R,
Grimm PC, Cheang M, Trpkov K, Solez K, Jeffery J. Beneficial effects of
treatment of early subclinical rejection: A randomized study. J Am Soc
Nephrol 1998;11: 2129‑2134.
Rush D, Nickerson P, Jeffery J, McKenna
R, Grimm P, Gough J. Protocol biopsies in renal transplantation: Research
tool or clinically useful? Current Opin Nephrol and Hypertens
1998;7:691‑694.
Immune Parameters Correlating Hyporepsonsiveness
Ronald
H. Kerman, Ph.D.
Long‑term renal allograft survival
is due to the efficacy of immunosuppressants or to an immunoregulatory
recipient (recip) hyporesponsiveness.
In vitro immunologic evaluation parameters were used to identify
immunologically low‑risk allograft recips with improved
long‑term graft survival. Recips
whose pretransplant (Tx) sera had little IgG anti‑HLA class I
antibody (<10 percent PRA, ELISA‑developed) experienced a 35
percent versus a 70 percent rejection frequency (p<0.01) and an 85
percent versus 74 percent 1‑year graft survival (p<0.01) when
compared with recips with reactive anti‑HLA sera (PRA >10
percent). The pre‑Tx
PRA sera <10 percent delineated an unsensitized, weak immune responder. The recip‑donor mixed lymphocyte reaction (MLR) also
served as an in vitro correlate, reflecting recip antidonor
hyporesponsiveness or hyperresponsiveness.
Hypo‑MLR recips experienced only 27 percent versus 54 percent
rejection episodes (p<0.05) and had a 92 percent versus 79 percent
1‑year graft survival (p<0.01) compared with hyper‑MLR
recips (SI >10). There was
significant correlation between recips with a pre‑Tx PRA sera <10
percent, and a post‑Tx hypo‑MLR: 89 percent (46/52) of
post‑Tx hypo‑MLR versus 19 percent (12/63) of hyper‑MLR
responders displayed pre‑Tx PRA sera <10 percent (p<0.001).
These data suggest that unsensitized recips (PRA <10 percent) may
develop an immunoregulated status resulting in donor hyporesponsiveness
and improved graft survivals and may be candidates for tapering and/or
withdrawing immunosuppressants.
Key
References
Kerman RH, Katz SM, Schoenberg L, Baraket
O, Van Buren CT, Kahan BD. Ten‑year follow‑up of mixed
lymphocyte reaction‑hyporesponsive living related cyclosporine
montherapy‑treated renal allograft recipients. Transplant Proc
1997;29:198‑199.
Kerman RH, Susskind B, Buelow R, Regan J,
Pauletty P, Williams J, Gerolami K, Kerman DH, Katz SM, Van Buren CT,
Kahan BD. Correlation of WLISA‑detected IgG and IgA anti‑HLA
antibodies in pretransplant sera with renal allograft rejection.
Transplantation 1996; 62:201‑205.
Kerman RH, Susskind B, Katz SM, Van Buren
CT, Kahan BD. Postrenal transplant MLR hypo‑responders have fewer
rejections and better graft survival than MLR hyper‑responders.
Transplant Proc 1997;29:1410‑1411.