Subclinical Rejection: The Sword of Damocles in Renal Transplantation?

John Lyons, PharmD

PGY-2 Solid Organ Transplant Pharmacy Resident

University Health System, San Antonio, TX

Division of Pharmacotherapy, The University of Texas at Austin College of Pharmacy

Pharmacotherapy Education and Research Center

University of Texas Health Science Center at San Antonio

January 13th

, 2016

Learning Objectives:

1. Describe the pathophysiology of renal transplant rejection

2. Define subclinical rejection and the role of the protocol biopsy in renal transplantation

3. Review current literature regarding the utility of treatment of subclinical rejection in renal

transplant recipients

4. Develop an evidence-based recommendation regarding the treatment of subclinical rejection

in renal transplant recipients

Lyons | { PAGE }

RENAL TRANSPLANTATION AND MAINTENANCE IMMUNOSUPPRESSION1-6

A. Renal transplantation1

a. Treatment of choice for end stage renal disease

b. Substantial benefit in comparison to long-term dialysis

i. Mortality

ii. Quality of life

iii. Cost

B. Goals of immunosuppression1,2

a. Prevent acute rejection of allograft

b. Prevent early graft loss

C. 1-year graft rejection has decreased and graft survival has increased steadily since the

1960s, correlating with development and release of new immunosuppressive agents2-6

Figure 1. 1-year graft survival and rejection rates, 1960-20133

D. Despite advances in immunosuppression and an impressive decrease in rejection rates

within the first year post-transplant, long-term renal allograft survival has not

significantly benefited3

a. Graft failure still remains roughly 4% per year

b. Chronic rejection is the second leading cause of graft failure

c. Acute rejection is an infrequent cause of graft failure

d. Epidemiologic studies have found a strong correlation between acute rejection

episodes and development of chronic allograft damage and allograft failure

0

10

20

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' 6 5 ' 7 0 ' 7 5 ' 8 0 ' 8 3 ' 8 5 ' 9 4 ' 9 5 ' 9 6 ' 9 8 ' 9 9 ' 0 0 ' 0 4 ' 0 8 ' 1 0 ' 1 2

1 year graft survival 1 year rejection rate

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e. Number, severity, and type of acute rejection episodes are strongly correlated

with early graft failure

Figure 2. Long-term Renal Transplant Graft Survival3

RENAL ALLOGRAFT REJECTION1-2, 7-9

A. Graft rejection may be cellular and/or humoral1-2,, 7-9

a. Although both pathways are intertwined, damage to the graft is dependent on cell

pathway orchestrating the immune response

B. Cellular graft rejection – “three signal model” of alloimmune responses1,2,9

a. Antigen-presenting cells (APC) identify non-self in graft and process graft antigen

for presentation

i. Engage alloantigen-reactive T-cells and memory T-cells

b. Signal 1

i. Antigen processed and presented by APC triggers T-cell activation

through T-cell receptor

c. Signal 2

i. Also known as “co-stimulation”

ii. Combination of signal 1 and 2 triggers signal transduction pathways that

lead to production of important inflammatory molecules

1. Interleukin-2

2. Tumor necrosis factor alpha

iii. These molecules play a role in activation of signal 3

d. Signal 3

i. Trigger for T-cell proliferation

ii. Leads to

1. Nucleotide synthesis

2. Differentiation of T-cells into various effector T-cells

e. Effector T-cells and graft damage

i. Effector T-cells emerge from lymphoid organs and orchestrate

inflammatory response

0

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0 2 4 6 8 10 12

Gra

ft S

urv

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Years Post Transplant

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ii. Infiltrate graft

iii. Activates several cell lines, including macrophages, B cells, plasma cells,

and chemokines

iv. Typical graft damage involves

1. Infiltration of the kidney tubules by mononuclear cells and intima

of small arteries

2. Direct cell lysis from cytotoxic T-cells and activated effector cells

C. Humoral graft rejection1,7-9

a. Also commonly known as antibody-mediated rejection (AMR)

b. Antigen presented to B cells, which differentiate and mature into active antibody-

producing plasma cells

c. Antibodies produced by plasma cells mark cells for destruction

i. Activates complement cascade

1. Binding of C1q to antigen-antibody complex on graft endothelium

triggers activation of complement cascade

2. End-product is membrane attack complex (MAC) which attacks

and destroys integrity of phospholipid bilayer of cells leading to

cell death

ii. Forms immune complexes which target cell for destruction via

macrophages or natural killer cells

d. Transplant recipients may develop donor-specific antibodies (DSA) to donor

human leukocyte antigen (HLA)

D. Types of rejection in renal transplantation1-2

a. Hyperacute rejection

i. Occurs when recipient’s immune system immediately rejects the donor

organ

ii. Typically occurs upon reperfusion of the newly transplanted organ, but

can be delayed up to 3 days post-transplant

iii. Characterized by rapid, widespread vascular thrombosis affecting arteries,

arterioles, and glomeruli

iv. With the development of detailed HLA phenotyping techniques and

donor-recipient cross-matching, incidence of hyperacute rejection is rare

b. Acute rejection

i. Most commonly refers to acute cellular rejection (ACR)

ii. Can occur at any time post-transplant

iii. 2 predominant forms

1. Cellular rejection (most common)

2. Humoral rejection

iv. Cellular rejection

1. Most common form of acute rejection

2. T-cell-mediated

3. Inflammatory infiltrates typically diminish rapidly upon successful

treatment, but edema, tubular inflammation, and tubular cell

damage may persist

v. Acute humoral rejection

1. Mediated by antibody-producing plasma cells

Lyons | { PAGE }

2. Diverse histologic appearance

3. Characterized by diffuse peritubular capillary staining for

complement component C4d, although not necessary

a. Linked to presence of DSAs and humoral rejection

CHRONIC REJECTION1, 10-12

A. Terminology and definition1, 10-12

a. Previously referred to as chronic allograft nephropathy (CAN)1

b. Due to presence of both alloimmune and non-alloimmune mechanism of

progressive graft injury, the terms “chronic rejection” and “chronic allograft

nephropathy” have been largely replaced by “interstitial fibrosis and tubular

atrophy” (IFTA)1

i. Now commonly referred to as CAN/IFTA

c. Progressive decline in allograft function gradually leading to allograft failure10-12

i. Multi-factorial

ii. Involves immunologic as well as non-immunologic factors

1. Immunologic

a. Cellular rejection

b. Humoral rejection

c. Medication non-adherence

2. Non-immunologic

a. Delayed graft function (DGF)

b. Infection

c. Hypertension

d. Post-transplant diabetes mellitus

e. Calcineurin inhibitor (CNI) toxicity

iii. Second leading cause of graft loss

d. Characterized by1, 11

i. Patch interstitial fibrosis with infiltrates of lymphocytes, plasma cells, and

mast cells

ii. Tubular atrophy or tubular dropout

iii. Arterial wall thickening with intimal fibrosis

iv. Glomeruli are often abnormal, constituting a lesion known as chronic

transplant glomerulopathy

Lyons | 6

Table 1. Risk Factors for the Development of CAN/IFTA10,12

Donor Derived Recipient Derived Deceased donor kidney

Non-heart beating donor kidney

Donor age > 60

Female Donor

Donor with prior cardiac history or vascular disease

Cold ischemic time

DGF

Obesity

Polyomavirus nephropathy

CNI toxicity

Recurrent renal disease or de novo glomerulopathy

Hypertension

Hyperlipidemia

Proteinuria

Diabetes

Medication non-compliance

HLA mismatch

Recipient pre-sensitization/panel reactive antibody

(PRA)

Presence of donor specific antibody (DSA)

Acute rejection

Subclinical rejection

TREATMENT OF REJECTION7-8, 10,13

A. KDIGO Guideline for the Care of Kidney Transplant Recipients: Treatment for Rejection

(2009)13

a. Cellular Rejection

i. First line, mild-moderate: High-dose or “pulse-dose” corticosteroids

ii. Steroid-resistant rejection or severe rejection: Rabbit antithymocyte

globulin

b. Humoral Rejection7-8, 13

i. Treatment is targeted at removing circulating DSA, B-cells, and antibody-

producing plasma cells

ii. Plasmapheresis and intravenous immune globulin (IVIG) serve as the

backbone for antibody-mediated rejection treatment

1. Rapidly removes circulating DSA

2. IVIG replaces IgG that is removed during the plasmapheresis

process, and is believed to have an effect on decreasing and

preventing rebound DSA production

iii. Targeted antibody therapies can be used in conjunction with

plasmapheresis and IVIG, with or without corticosteroids

1. Rituximab

2. Bortezomib (not included in 2009 KDIGO Guidelines)

3. Eculizumab (not included in 2009 KDIGO Guidelines)

c. Chronic rejection10

i. No specific treatment modality has been identified

ii. The best “treatment” for chronic rejection/IFTA is prevention

1. Prompt diagnosis and treatment of acute rejection episodes

Lyons | { PAGE }

2. Management of post-transplantation hypertension, hyperlipidemia,

diabetes, and proteinuria

3. Optimization of immunosuppressive medications, especially CNIs

4. Maintenance of medication adherence

THE PROTOCOL BIOPSY14-25

A. Definition14-23

a. Biopsy performed at pre-specified post-transplant milestones regardless of

allograft function

b. Surveillance in nature

c. Performed in less than 20% of transplant centers in the U.S.26

B. Serum creatinine: an old friend that’s not keeping up24-25

a. Serum creatinine and proteinuria lack sensitivity as measurement for kidney

dysfunction24

i. Although widely used due to its ease of measurement and relatively low

cost, using serum creatinine as measure of glomerular filtration rate (GFR)

has several disadvantages

ii. Serum creatinine has wide inter-patient variability with regard to

1. Sex

2. Age

3. Race

4. Muscle mass and body weight

iii. Change in serum creatinine may not manifest until underlying pathology is

advanced

1. Wide inter-assay variability exists between labs25

b. Entities such as early acute rejection and recurrent focal segmental

glomerulosclerosis develop within hours to days post-transplant and become

clinically apparent early26

i. All will present as an increase in serum creatinine

ii. Using serum creatinine as primary marker lacks specificity for the

pathologic etiology

c. Other causes of graft dysfunction develop over a longer period of time26,27

i. Can be preceded by a “subclinical phase”

ii. Changes and damage to graft can elude routine laboratory monitoring

d. Other markers, with greater sensitivity and specificity for monitoring graft

function, have been proposed24,26

i. Urinary biomarkers such as cystatin c

ii. Serial 24-hour urine creatinine collections and GFR monitoring

iii. Protocol biopsies

iv. None of these methods are without limitations

C. Advantages of protocol biopsies14-23

a. Detect incidental findings

i. BK virus nephropathy

ii. CNI toxicity

iii. Chronic rejection

iv. Primary disease recurrence

Lyons | { PAGE }

v. De novo glomerulonephritis

vi. Asymptomatic urinary tract infections

b. Identify early clinical rejection

c. Identify subclinical rejection (SubR)

d. Provide a more useful marker than serum creatinine alone in monitoring graft

function and changes over time in high immunologic risk renal transplant

recipients

i. Identification of early subclinical rejection on protocol biopsy has allowed

for the opportunity for prompt treatment and modification of maintenance

immunosuppression to prevention of progression28

D. Disadvantages of protocol biopsies14-23

a. Invasive

b. Risk of complications

i. Hematuria

ii. Perinephric hematoma

iii. Bowel perforation

iv. Vasovagal reaction

v. Graft loss

c. Sampling error

d. Inter- and intra-observer variability among pathologists

e. Cost

f. Patient quality of life

SUBCLINICAL REJECTION26-46

A. Definition/Epidemiology26-27, 29-33

a. Presence of histological features of acute rejection on renal biopsy in absence of a

decline in renal function

b. Clinically significant decline in renal function - increase in serum creatinine ≥ 10-

25% of baseline29

c. Prevalence is highest early post-transplant

i. Most common in first 6 months post-transplant

ii. One of the earliest studies evaluating subclinical rejection reported an

incidence of 30% within the first 3 months of transplant in the era of

cyclosporine (CsA)-based immunosuppression32

iii. Declines to 18% at 12 months post-transplant

iv. Incidence of subclinical rejection in the era of tacrolimus (TAC)-based

immunosuppression has declined tremendously

1. 6-8% within 1-3 months post-transplant33

B. Pathophysiology26, 34-36

a. Alloimmune process similar to clinical acute rejection

b. Key differences and similarities between subclinical and clinical rejection

i. Macrophage activation marker allograft inflammatory factor-1 found to be

10-fold higher in patients with clinical rejection

1. Detrimental effect on renal function in clinical rejection may be

due to a vasoconstrictive or cytotoxic substances produced by

macrophages

Lyons | { PAGE }

ii. Reduced chemokine, cytokine, and cytotoxic lymphocyte product amounts

in comparison to clinical rejection

C. Risk factors37-39

a. HLA mismatch

i. Class II HLA antigen mismatch (DR, DP, DQ) more closely associated

with subclinical rejection in comparison to Class I HLA antigen mismatch

(A, B, C)

b. Prior sensitization

i. Blood transfusion

ii. Pregnancy

iii. Previous organ transplant

iv. Infection

c. Previous clinical rejection

d. Induction immunosuppression with interleukin-2 receptor antagonists (IL-2RA)39

D. Correlation to acute and chronic rejection35, 40

a. Major clinical concern surrounding subclinical rejection pertains to potential

implications on long-term graft damage and survival

i. Previous literature demonstrated that a cohort of patients with a 7-day

post-transplant biopsy showing subclinical rejection go on to develop

clinical rejection if untreated40

ii. Of patients with borderline changes on surveillance biopsy, 20%

experienced acute rejection within 6 months in a single-center study40

iii. Subclinical rejection found on 3-month protocol biopsy was a positive

predictor of interstitial fibrosis at 1 year post-transplant35

E. A new picture of graft damage26-27

a. With developing characterization of subclinical rejection and implications

regarding acute and chronic rejection, there may be more that contributes to

progressive allograft damage than previously speculated

b. Subclinical rejection may represent a “behind the scenes” pathology that

contributes to chronic rejection and acute rejection episodes

F. Treatment29-33

a. In the era of CsA-based maintenance immunosuppression, a single study has

demonstrated benefit with early treatment of subclinical rejection on protocol

biopsy32

i. Prospective, randomized trial of 72 patients demonstrated that patients

who received early protocol biopsies and pulse steroids had significantly

lower serum creatinine and higher GFR at 2 years post-transplant in

comparison to patients who did not receive protocol biopsies

ii. Patients randomized to protocol biopsies also had a lower incidence of

clinical rejection episodes at 2, 3, and 7-12 months post-transplant

iii. Patients in protocol biopsy group also had significantly lower chronic

interstitial and tubular changes at 6 months post-transplant

b. As incidence of subclinical rejection has declined significantly in the era of TAC-

based maintenance immunosuppression regimens, benefit of treating subclinical

rejection has been difficult to demonstrate33

Lyons | { PAGE }

G. Subclinical rejection in the TAC era Study

46 Rush D, Arien D, Boucher A, et al. Lack of benefit of early protocol biopsies in

renal transplant patients receiving TAC and MMF: a randomized study. Am J

Transplant. 2007; 7: 2538-45.

Design Open-label, randomized, prospective, multi-center, parallel-group study in 11

centers in Canada and 1 center in the US

Objective Assess the effect of treating subclinical rejection on protocol biopsy within the first

3 months post-transplant

Interventions Randomized 1:1 to:

Protocol biopsies at 1, 2, 3, and 6 months (Biopsy Arm, BA)

Biopsies at 6 months only (Control Arm, CA)

All patients received TAC, mycophenolate mofetil (MMF), and prednisone for

maintenance immunosuppression

All clinical and subclinical rejection episodes were treated with a 2-week tapering

course of prednisone starting at 200 mg, with the option for investigators to use

methylprednisolone and anti-lymphocyte agents at their discretion

Endpoints Primary: Prevalence of chronic histology at 6 months

Secondary:

Prevalence of subclinical rejection at 6 months

Frequency of biopsy-confirmed or suspected acute rejections within months

0-6

Renal function at 6 months post-transplant

Results ITT Patient Population: 218 patients included (111 in BA, 107 in CA)

Mean age 47.7 years, 67-71% male, 73.8-79.3% Caucasian

No significant (NS) difference between arms with regard to baseline

characteristics

Primary: NS difference in chronic histology scores between both groups (p = 0.09),

although both experienced significant increases in scores between implantation and

month 6

Secondary: Prevalence of SubR at 6 months 9% in the BA vs. 6% in the CA, p =

0.48

Acute rejection rate NS different between arms, with 12 episodes in the BA

and 8 episodes in the CA, respectively (P = 0.44)

NS difference in mean serum creatinine and estimated creatinine clearance

between arms

Author

Conclusions There is no benefit to the procurement of early protocol biopsies in renal

transplant patients receiving TAC, MMF, and prednisone, at least in the

short term, likely due to the low prevalence of SubR

Critique Strengths:

All patients treated with TAC-

based immunosuppression

Multi-center, randomized,

controlled trial

Limitations:

Small sample size

Lack of true control group

Lack of long-term follow-up

Bottom Line In the era of TAC-based immunosuppression, there may be a lower incidence of

subclinical rejection than previously reported in CsA-based regimens. More frequent

protocol biopsies do not prevent subclinical rejection at 6 months. This calls into

question utility of more frequent surveillance biopsies to detect subclinical rejection,

let alone whether or not it should be treated.

Lyons | { PAGE }

CLINICAL QUESTION: IS IT WORTHWHILE TO TREAT SUBCLINICAL

REJECTION?

CLINICAL DATA47-49

Study

47 Kee TYS, Chapman JR, O’Connell PJ et al. Treatment of subclinical rejection

diagnosed by protocol biopsy in kidney transplants. Transplantation. 2006; 82: 36-

42.

Design Retrospective analysis of kidney transplant recipients at a single center between

01/01/2001 and 12/31/2003

Objective Assess the effect of treating subclinical rejection on protocol biopsy within the first

3 months post-transplant

Population Inclusion:

Adult kidney transplant recipient

Combined kidney-pancreas

transplant recipient

Protocol biopsies performed at 1

and 3 months post-transplant

CNI-based immunosuppression

for the first year post-transplant

Exclusion:

Both 1 and 3 month protocol

biopsies not performed

Participation in a clinical drug

trial

Loss to follow-up within 3

months post-transplant

Non-CNI-based maintenance

immunosuppression

Graft loss within first 3 months

post-transplant

Interventions Maintenance immunosuppression

Kidney transplant recipients

o CsA microemulsion 10 mg/kg/day (initial) or TAC 0.2 mg/kg/day

(initial), then adjusted to center goal levels

o MMF 2 gm/day

o Prednisolone 20 mg/day for 3 months, then tapered to 10 mg daily

by 6 months

Kidney-pancreas transplant recipients

o CsA microemulsion 10 mg/kg/day or TAC 0.2 mg/kg/day adjusted

to center goal levels

o MMF 3 gm/day

o Prednisolone 30 mg/day for 3 months, then tapered to 10 mg daily

by 6 months

Treatment of subclinical rejection:

Intravenous methylprednisolone 500 mg daily x 3 days OR increase of

prednisolone dose to 1 mg/kg/day x 5 days (no maximum dose), then

tapered down to baseline dose

OR

Increase in overall immunosuppression (IMS)

o Increase in baseline CsA or TAC dose

o Increase in MMF dose

o Switching CsA to TAC

Endpoints Subclinical rejection at 1 or 3 months post-transplant, defined as histological

evidence of acute rejection or borderline changes in patients with stable renal

function (< 25% change in serum creatinine from baseline)

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Subclinical rejection stratified:

Acute (A-SubR) – at least Banff grade 1A

Borderline (B-SubR) – foci of mild tubulitis and at least 10-25%

mononuclear cell interstitial inflammation of the cortex

Results Patient Population:

88 patients included (59 kidney transplant alone, 29 kidney-pancreas)

Mean age 41.3 ± 12.1 years, 57.9% male

45 patients received CsA-based immunosuppression vs. 43 TAC-based

Subclinical Rejection:

46.6% overall incidence of subclinical rejection (n = 41/88)

o A-SubR: 12.5% (n = 11/88)

o B-SubR: 34.1% (n = 30/88)

o Of the 41 episodes of SubR, 33 were identified during the protocol-

defined endpoints, and 8 were identified upon repeat biopsy

25% and 10.2% of SubR was identified at 1 month and 3 months post-

transplant, respectively

SubR occurred in 17 CsA-treated patients and 24 TAC-treated patients

Treatment of Primary Subclinical Rejection Episodes (n = 33)

1 month

(n = 22)

3 months

(n = 9)

12 months

(n = 2)

Increase in IMS alone 4 0

CsA TAC alone 1 2

Pulse steroids alone 2 1

Pulse steroids AND

Increased

IMS

CSATAC

OKT3

10

1

1

1

1

No Treatment 3 5 1

Progression of Subclinical Rejection

Early chronic graft damage was observed in 29.5% (n = 26/88) of all 1-

month protocol biopsies

Higher incidence of chronic graft damage in patients with SubR on 1-month

protocol biopsy (p < 0.005)

Increased IFTA scores in biopsies with SubR vs. no SubR (p < 0.05) at 1

month post-transplant

By 3 month protocol biopsy, inflammation had largely decreased in

comparison to 1 month protocol biopsies

o Significantly lower Banff scores (p < 0.05)

o Chronic allograft damage was increased in the cohort of patients

who were not treated for 1 month SubR compared to those who

were treated (p < 0.05)

Author

Conclusions SubR was common in the study cohort and associated with early chronic

tubulointerstitial damage

Histological progression of injury appeared to be stabilized by treatment

with pulse corticosteroids combined with augmentation of baseline

immunosuppression

Critique Strengths:

Included patients with TAC-

Limitations:

Small sample size

Lyons | { PAGE }

based immunosuppression

Wide variety of methods for

treating subclinical rejection,

allowing for physician freedom

within protocol

Retrospective analysis

Pilot study, observational in

nature

No stratification of results based

on CNI used or organs

transplanted

Wide variety of treatment

options make interpretation and

application of a similar protocol

difficult

No subgroup analysis of risk

factors for subclinical rejection

Bottom Line Treatment of subclinical rejection has impact on the progression of chronic

damage within the renal allograft

Optimal time for protocol biopsy for detection of subclinical rejection is

within 3 months post-transplant

SubR incidence at 1 month post-transplant suggest benefit of protocol

biopsies earlier on in the post-transplant course than 3 months

The optimal treatment regimen for SubR has yet to be elucidated

Study

48 Szederkenyi E, Ivanyi B, Morvay Z, et al. Treatment of subclinical injuries detected

by protocol biopsy improves the long-term kidney allograft function: a single center

prospective randomized clinical trial. Transplantation Proceedings. 2011; 43: 1239-

43.

Design Single center, prospective, randomized trial

Objective Evaluate benefit of early and late protocol biopsies

Evaluate the impact of early treatment of discovered pathologies on allograft

function at 2 and 3 years and long-term (5-year) graft survival

Population Inclusion:

Age > 18 years

Stable graft function at 3 months post-transplant (serum creatinine < 3.39

mg/dL)

No clinical symptoms of rejection within 1 month prior to randomization

Maintenance CNI and MMF-based immunosuppression

Stable immunosuppressive trough levels

o TAC 5-15 ng/mL

o CsA 100-250 ng/mL

Good compliance to treatment

Interventions Patients randomly assigned under controlled allocation 1:1 to two biopsy regimens:

Protocol biopsies at 3 and/or 12 months (biopsy group) within 1 of 3

protocols:

o Protocol biopsy at 3 months only

o Protocol biopsy at 12 months only

o Protocol biopsies at both 3 and 12 months

Clinical surveillance only, no protocol biopsy (control group)

Subclinical rejection: defined as histologic findings consistent with the occurrence

of an acute rejection episode without associated graft dysfunction

Treated with a steroid pulse therapy

Endpoints Outcomes assessed include:

Lyons | { PAGE }

Graft function at 3 months, 1 year, and annually up to 5 years post-

transplant, measured as estimated GFR (eGFR) according to the Cockroft-

Gault method an the Modification of Diet in Renal Disease (MDRD)

formula

Descriptive results of protocol biopsies

Safety of protocol biopsy as defined by incidence of biopsy-related adverse

events

Results Patient Population:

Five-year follow-up data available on 145 patients (N=113 protocol biopsy,

N=51 control)

Majority male (56% and 58% in protocol biopsy and nonbiopsy groups,

respectively), mean age 44 years

NS difference in baseline characteristics, including HLA mismatch,

proportion of patients with PRA ≥ 50%, and induction therapy used

Patients randomized 1:1 to TAC:CsA within groups for maintenance

immunosuppression

o Protocol biopsy group: 59 TAC, 53 CsA

o Control group: 25 TAC, 24 CsA

Graft Function:

NS difference in eGFR at 1 and 2 years post-transplant between groups

Significantly lower serum creatinine (1.80 ± 0.51 mg/dL in protocol biopsy

group vs. 2.45 ± 1.05 mg/dL in control group, p=0.003) and greater eGFR

observed in protocol biopsy arm (46.0 ± 13.8 mL/min/1.73 m2 in protocol

biopsy group vs. 35.4 ± 15.0 mL/min/1.73 m2 in control group, p = 0.002) at

3 years post-transplant

Subclinical Rejection

65 cases of subclinical acute rejection identified by 1 year post-transplant all

treated with pulse steroids

Numerically higher amount of SubR identified at 12 months post-transplant,

but not statistically significant (p = 0.08)

Safety:

7 patients experienced biopsy-related complications

o All patients were discharged after the 4-hour post-biopsy

observation time

Author

Conclusions Protocol biopsy is an excellent method for the early diagnosis of disorders in

the transplanted kidney and to monitor the effects of immunosuppression

The protocol biopsy, followed by appropriate treatment, promotes

preservation of kidney allograft function and therefore improves long-term

graft survival

Critique Strengths:

Prospective, randomized study

All subclinical rejection

episodes treated the same, with

pulse steroid therapy

Long-term follow-up

Inclusion and randomization of

patients to TAC and CsA

Limitations:

Single center, small sample size

No definition of graft

dysfunction

Lack of pre-defined outcomes

No stratification based on CNI

No stratification of results based

on protocol biopsy regimen used

“Pulse steroids” is ambiguous

Bottom Line Use of protocol biopsies within the first year post-transplant, as well as

Lyons | { PAGE }

prompt treatment of subclinical rejection has beneficial effect on long-term

(> 2 years) renal graft function

o There appears to be no beneficial effect on allograft function at 1

year post-transplant

Study

49 Gigliotti P, Lofaro D, Leone F, et al. Early subclinical rejection treated with low

dose i.v. steroids is not associated to graft survival impairment: 13 years’ experience

at a single center. J Nephrol. 2016; 29: 443-9.

Design Single center, retrospective study

Objective Evaluate the incidence of early subclinical rejection revealed by protocol biopsy at 1

months post kidney transplantation, factors potentially involved in the manifestation

of SubR, and evaluate the association between SubR previously treated with low-

dose intravenous steroids and long-term graft survival

Population Inclusion:

Primary kidney transplant

Deceased donor

Protocol biopsy at day 30 post-

transplant

Exclusion:

Primary non function

For-cause biopsy before day 30

post-transplant

Interventions SubR defined as absence of functional deterioration and with presence of histologic

findings indicative of rejection on the basis of tubulitis (t) and mononuclear cell

infiltration (i) scores:

t score ≥ 1 and i score > 0 in the absence of functional deterioration (< 15%

decrease in serum creatinine) classified as SubR

Immunosuppression regimens:

Induction: basiliximab

Maintenance:

o CNI (TAC or CsA) adjusted to levels:

TAC trough levels of 5-15 ng/mL

CsA C0 levels of 150-300 ng/mL

CsA C2 levels of 1400-1800 ng/mL early post-transplant

and 800-1200 ng/mL later after transplant

o MMF 1.5 gm/day

o Steroids

Treatment:

SubR: methylprednisolone 250 mg/day intravenous for 3 days

Acute rejection: methylprednisolone 500 mg/day intravenous for 3 days

o Anti-thymocyte globulin was used as clinically and histologically

indicated

Patients divided into normal, SubR, and acute rejection groups according to Banff

classification on protocol biopsy for analysis

Endpoints 10-year graft survival

Cox-proportional hazards regression model to determine factors associated

with graft survival

Results Patient population:

Italian

174 patients screened, 159 patients underwent protocol biopsy

59.8% male, mean age 46.15 ± 12.2 years

69 patients (39.7%) experienced DGF requiring ≥ 1 days on dialysis

113 of 174 patients received TAC as CNI (64.9%)

Lyons | { PAGE }

Outcomes of interest:

Protocol biopsy results (n =159)

o Normal histology: 142 patients (89.3%)

o SubR: 17 patients (10.7%), all treated with pulse steroids per

protocol

10 patients with functional changes

o 2 with CsA toxicity

o 8 with acute rejection

NS difference in SubR between patients treated with TAC vs. CsA (9.8%

treated with TAC vs. 9.7% treated with CsA, p = 0.202)

Factors associated with SubR (multivariate analysis):

o Donor age: OR 1.04 (95% CI 1.01-1.09)

o DGF: OR 1.08 (95% CI 1.03-1.12)

10-year graft survival:

o NS difference between SubR group and normal histology group

o Acute rejection group had a significantly lower 10-year graft

survival in comparison to both SubR and normal histology groups

o Factors associated with 10-year graft failure (multivariate analysis):

Donor age: HR 1.03 (95% CI 1.01-1.05)

DGF: HR 1.57 (95% CI 1.04-2.22)

Acute rejection: HR 5.22 (95% CI 1.70-16.01)

Subclinical rejection WITH TREATMENT was not

independently associated with 10-year graft failure

Author

Conclusions

An early protocol biopsy performed 1 month after renal transplantation is a useful

tool to detect subclinical rejection, and anti-rejection treatment for SubR with low-

dose i.v. pulse steroids could be an appropriate strategy to improve kidney transplant

graft survival in the long term

Critique Strengths:

All patients with SubR received

identical treatment

Greater proportion of patient

population received TAC,

mimicking current clinical

practice

Long-term follow up

Limitations:

No evaluation of graft function

outside of overall graft survival

Single-center, Italian patient

population hinders external

validity

Retrospective

No true control group

Bottom Line Results support the practice of using early (< 3 month post-transplant)

protocol biopsies to evaluate early subclinical changes in renal transplant

recipients

Standardized treatment of subclinical rejection with low-dose pulse steroids

and lack of significant difference in graft survival at 10-years post-transplant

suggests that pre-emptive treatment is able to attenuate or even reverse these

subclinical changes

Patients with treated subclinical rejection behave more like patients without

rejection

A prospective, randomized trial would be the gold-standard to assess this

hypothesis

Lyons | { PAGE }

SUMMARY

Incidence of SubR was drastically reduced with the introduction of TAC and MMF-based

maintenance immunosuppression regimens

Few well-designed studies have directly evaluated the benefit of treatment of SubR

The optimal time for detection of SubR is within the first 3 months post-transplant

Early treatment of SubR has negligible benefit on renal function at 1 year post-transplant,

but is associated with significant benefit on long-term (> 2 year) renal function

Pulse steroids are able to attenuate and reverse SubR episodes

RECOMMENDATION

Patients at high immunologic risk for the development of CAN/IFTA should be

monitored with protocol biopsies post-transplant

o Positive crossmatch

o ABO-incompatible

o Delayed graft function

For centers utilizing protocol biopsies after renal transplant, biopsies should be performed

within 3 months post-transplant

For centers performing routine protocol biopsies within the first year post-transplant,

SubR should be treated if detected

o Methylprednisolone 250-500 mg daily for 3 days (similar to treatment for acute

rejection)

o Optimization of immunosuppression

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Appendix A. Abbreviations

ACR: Acute cellular rejection

AMR: Antibody-mediated rejection

APC: Antigen-presenting cell

A-SubR: Acute subclinical rejection

B-SubR: Borderline subclinical rejection

CAN: Chronic allograft nephropathy

CNI: Calcineurin inhibitor

CsA: Cyclosporine

DGF: Delayed graft function

DSA: Donor specific antibody

eGFR: Estimated glomerular filtration rate

GFR: Glomerular filtration rate

HLA: Human leukocyte antigen

IFTA: Interstitial fibrosis and tubular atrophy

IL-2RA: Interleukin 2 receptor antagonist

IVIG: Intravenous immune globulin

MAC: Membrane attack complex

MMF: Mycophenolate mofetil

SubR: Subclinical rejection

TAC: Tacrolimus

Appendix B. Banff ’97 Classification of Renal Allograft Pathology, 2009 Update9

Diagnosis Histologic Characteristics and Grading

Normal Normal histology of biopsy sample

Borderline Changes

“Suspicious” for acute t-cell mediated

rejection:

1. No intimal arteritis

2. Foci of tubulitis with minor interstitial

infiltration

Interstitial infiltration with mild tubulitis

Acute T-cell-mediated rejection

IA. Interstitial inflammation (> 25% of

parenchyma) and foci of moderate tubulitis

IB. Interstitial inflammation (>25% of

parenchyma) and foci of severe tubulitis

IIA. Mild to moderate intimal arteritis

IIB. Severe intimal arteritis comprising >25%

of luminal area

III. Transmural arteritis and/or arterial fibrinoid

change and necrosis of medial smooth muscle

cells with accompanying lymphocytic

inflammation