Supplementary Materials for

Individualizing liver transplant immunosuppression using a phenotypic

personalized medicine platform

Ali Zarrinpar,* Dong-Keun Lee, Aleidy Silva, Nakul Datta, Theodore Kee,

Calvin Eriksen, Keri Weigle, Vatche Agopian, Fady Kaldas, Douglas Farmer,

Sean E. Wang, Ronald Busuttil, Chih-Ming Ho,* Dean Ho*

*Corresponding author. E-mail: azarrinpar@mednet.ucla.edu (A.Z.); chihming@g.ucla.edu (C.-M.H.);

dean.ho@ucla.edu (D.H.)

Published 6 April 2016, Sci. Transl. Med. 8, 333ra49 (2016)

DOI: 10.1126/scitranslmed.aac5954

This PDF file includes:

Subject clinical details

Methods

Fig. S1. The effect of cotrimoxazole and fluconazole dosing on patients’ trough

levels.

Fig. S2. PPD tacrolimus-prednisone interaction plots for patients PPD1, PPD2,

and C1.

Fig. S3. PPD tacrolimus-cotrimoxazole interaction plots for patient PPD4.

Fig. S4. Retrospective PPD-guided tacrolimus and prednisone dosing

optimization for patients C1 and C2.

Table S1. Clinical summaries of control patients (C1 to C4) and PPD-guided

patients (PPD1 to PPD4).

Table S2. Data used for statistical comparisons in Fig. 5.

Table S3. Patient-specific antibiotic and antifungal dosing changes and

corresponding tacrolimus trough levels.

Table S4. Patient-specific anti-inflammatory and immunosuppressant dosing

changes and corresponding trough levels.

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencetranslationalmedicine.org/cgi/content/full/8/333/333ra49/DC1)

Movie S1 (.mov format). Introduction to PPD and patient recalibration.

Movie S2 (.mov format). Patient PPD2 recalibration tutorial.

www.sciencetranslationalmedicine.org/cgi/content/full/8/333/333ra49/DC1

Movie S3 (.mov format). Patient PPD3 recalibration tutorial.

SUBJECT CLINICAL DETAILS

We enrolled 8 consecutive adult liver transplant patients and randomized to two groups:

physician-guided standard of care dosing (Control Patients: C1-C4) and PPD-guided dosing

(PPD1-PPD4) (table S1). Physician-guided standard of care dosing was used for the first 10 days

of tacrolimus dosing after liver transplant, during which all patients were transitioned from

methylprednisolone to prednisone based on an established clinical protocol. To avoid

confounding effects from this protocol and to allow the establishment of steady-state drug

concentrations, personalized tacrolimus dosing via PPD started after this period (after post-

transplant tacrolimus dosing day 10) for the four PPD-assisted patients, whereas the four control

patients stayed on the standard of care. Additional drugs were given to each patient during the

course of treatment due to their diverse range of pre- and post-operative conditions.

Control patients. C1 was a 50-year-old woman with nonalcoholic steatohepatitis (NASH)

cirrhosis. Prior to transplantation, she was in the hospital ICU for a few days because of acute

decompensation. Her MELD (model of end-stage liver disease) score at transplantation was 36.

The MELD score predicts patient mortality within three months without transplantation, and

MELD values range from 6 (lowest acuity) to 40 (highest acuity). Her peri- and post-operative

course was uncomplicated. She was discharged on post-operative day 22.

C2 was a 39-year-old man with alcoholic cirrhosis. He was in the hospital ICU for

approximately one month prior to receiving an organ offer. During that time he was

intermittently on the ventilator. He was on vasopressors and continuous dialysis before

transplantation and his MELD score was 40. His operative course was unremarkable, except for

the fact that his biliary anastomosis was stented with a T-tube. Post-operatively he required

prolonged ventilator support and the attendant inability to swallow. He was on dialysis after liver

transplantation until his discharge on post-operative day 46.

C3 was a 73-year-old man with a physiologic MELD score of 13 who came from home to

be transplanted for alcoholic cirrhosis and hepatocellular carcinoma. His operative course was

complicated by severe ischemia reperfusion injury and cardiac instability. He was temporarily

closed and biliary anastomosis was deferred until postoperative day 3. His post-operative course

was very complex. He initially had poor graft function with liver function tests that were

persistently elevated. Multiple explorations and biopsies led to equivocal results, with the poor

graft function being ascribed to persistent infection, antibody-mediated rejection [for which he

underwent plasmapheresis, intravenous immunoglobulin (IVIg) injection, and treatment with

bortezomib]. Ultimately, interventional radiology embolized a splenorenal shunt, and the patient

underwent endoscopic stenting of his biliary anastomosis resulting in improved hepatic function.

Fortunately, his renal function was unaffected during this time. He was discharged on post-

operative day 98.

C4 was a 61-year-old man with a physiologic MELD score of 19 who came from home to

be transplanted for hepatitis C cirrhosis and hepatocellular carcinoma. His operative course was

complicated by coagulopathy requiring deferment of the biliary anastomosis until post-operative

day 2. He suffered some acute kidney injury from the operation but did not require hemodialysis.

He was discharged on post-operative day 29.

PPD patients. PPD1 was a 53 year-old-man with alcoholic cirrhosis. He had been in the

hospital ICU on a ventilator and requiring vasopressors and continuous dialysis for months prior

to receiving an organ offer. His MELD score was 40 at the time of transplantation. His intra- and

post-operative course was mostly unremarkable. Because of his debility, he was unable to

swallow for weeks after his operation. His renal function has not yet recovered. He was

discharged on post-operative day 33.

PPD2 was a 56-year-old man with hepatitis C who came from home to be transplanted

with a MELD score of 25. His operative course was unremarkable. He had some acute kidney

injury after transplantation but that gradually recovered. His post-operative course was

complicated by a biliary leak that required drain placement and endoscopic biliary stent

placement. He was discharged on post-operative day 30.

PPD3 was a 65-year-old man with hepatitis C cirrhosis and hepatocellular carcinoma. He

came from home for an organ offer with a physiologic MELD score of 9. Intraoperatively he

suffered from stress-induced cardiomyopathy and needed to have his biliary anastomosis

deferred until post-operative day 3. His cardiac function improved slowly, and his perioperative

acute kidney injury recovered. He was discharged on postoperative day 22.

PPD4 was a 54-year-old morbidly obese woman with a MELD score of 40 who was

transplanted for NASH cirrhosis. She had been in the ICU for days because of encephalopathy

and need for continuous dialysis. Her operation was notable for the need to have a Roux-en-Y

biliary anastomosis due to scarring from previous operations. She had some persistent infections

requiring antibiotics. Her renal function recovered slowly after liver transplantation and she no

longer needs dialysis. She was discharged on post-operative day 33.

SUPPLEMENTARY METHODS

Prospective clinical treatment protocol

Following liver transplant, patients were started on a regimen of tacrolimus, MMF, and

methylprednisolone. According to the protocol, daily methylprednisolone dosing was tapered

using a 1000-150-210-120-80-40-20 mg regimen. Methylprednisolone was subsequently

changed to prednisone given at 20 mg daily which was subsequently tapered per protocol. MMF

dosing was performed according to the protocols determined by the clinical team based on the

recipient’s individual circumstances. Trough levels were taken between 4:00 and 6:00 AM daily,

and a first dose of tacrolimus was administered between 5:00 and 6:00 AM daily, following the

whole blood trough recording. The clinical team obtained the daily treatment regimen details

including drugs already administered, drugs to be administered, hemodialysis to be performed,

co-infections, and planned prophylaxis and sent an updated clinical chart for each patient to the

PPD team. Following PPD analysis, we sent the suggested total daily tacrolimus administration

information to the clinicians prior to the administration of the evening dose.

Dosing days, D(X), were defined based on specific parameters immediately following

transplantation. Commencing from the first day of tacrolimus dosing following transplantation,

the target range and immunosuppressant dosing levels were changed frequently according to

protocol. For example, methylprednisolone dosing would be introduced with tacrolimus dosing

and was tapered from 1000 to 0 mg within the first 5 days following transplantation, at which

time it was replaced with a prednisone taper beginning at 20 mg. PPD-assisted treatment

commenced after the transition from methylprednisolone to prednisone. D(1) of the control

group was defined as the average value of the starting day of the PPD-treated patients which was

10 days after tacrolimus was introduced into patient’s regimen.

Retrospective parabolic personalized dosing process

Two anonymized liver transplantation patients’ (C1 and C2) clinical data, such as trough level

and drug regimen dosages, were obtained for analyses for the retrospective study. In order to

recommend optimal dosages, a 2nd order polynomial fit for each patient was made from linear

regression with covariates: tacrolimus and prednisone. The original PPD calibration required a

minimum of 6 different dosage combinations. In this process, the inclusion of the two drugs

allowed for better representation of each patient’s data and examination of the effects of drug-

drug interaction within each patient’s regimen. The importance of the retrospective PPD is based

on its ability to simultaneously identify optimal drug dose combinations for tacrolimus and

prednisone at specific time points that enabled the trough levels to remain within the prescribed

target ranges. Prednisone was administered according to the Dumont-UCLA Liver Transplant

Center’s clinical guidelines, which depend on each patient’s risk for rejection, recurrent disease,

or complication from steroids. The center’s guidelines are provided for reference.

If the patient is diagnosed with autoimmune hepatitis (AIH), primary sclerosing

cholangitis (PSC), or primary biliary cholangitis (PBC), a standard steroid taper is provided (7

days). Patients are to receive 1000mg hydrocortisone on the way to or in the operating room

(OR) with the following schedule: Day 1: 50 mg every 6 hours (q6h) x 4 methylprednisolone;

Day 2: 40 mg q6h x 4 methylprednisolone; Day 3: 30 mg q6h x 4 methylprednisolone; Day 4: 20

mg q6h x 4 methylprednisolone; Day 5: 20 mg every 12 hours (q12h) x 2 methylprednisolone;

Day 6: 10 mg q12h x 2 methylprednisolone; Day 7: 20 mg prednisone and wean over 3 months.

If the patient is diagnosed with hepatitis B, hepatitis C, alcoholic liver disease or non-

alcoholic steatohepatitis (NASH), a rapid steroid taper is provided (5 days). Patients are to

receive 1000mg hydrocortisone on the way to or in the OR with the following schedule: Day 1:

50 mg q6h x 4 methylprednisolone; Day 2: 25 mg q6h x 4 methylprednisolone; Day 3: 25 mg

q12h x 2 methylprednisolone; Day 4: 10 mg q12h x 2 methylprednisolone; Day 5: 20 mg

prednisone and wean over 1 month.

If the patient is at high risk of steroid complications, a steroid-free immune suppression

protocol is implemented: Rabbit Anti-Thymocyte Globulin (RATG) 1.5 mg/kg over 6 hours

starting during anhepatic phase; 500 mg methylprednisolone iv premedication before first dose

of RATG to minimize cytokine release; RATG 1.5 mg/kg on post-transplant day 2;

Mycophenolate Mofetil (MMF) starting postoperative day (POD) 1: 1,000 mg twice a day (bid)

x 3 months. If the patient is diagnosed with PBC, PSC, or AIH, then MMF is administered

indefinitely. Tacrolimus is administered starting POD 3-7; goal 6-8 until week 12; goal 3-5 until

week 24; goal ~3 until week 52; goal 1-3 thereafter.

.

Fig. S1. The effect of cotrimoxazole and fluconazole dosing on patients’ trough levels. These

patients were given cotrimoxazole and fluconazole, and had pure correlations between the

dosage changes in these drugs and changes in tacrolimus trough levels. These pure correlations

were used to pre-emptively dose tacrolimus for days with anticipated regimen changes. (A)

Cotrimoxazole dosing (blue) and the trough levels (red) were plotted against the dates of

administration for C4, PPD1, and PPD4. (B) Fluconazole dosing (purple) and the trough levels

(red) were plotted against the dates of administration for C2, C4, and PPD4.

Fig. S2. PPD tacrolimus-prednisone interaction plots for patients PPD1, PPD2, and C1. 3D

drug interaction maps of tacrolimus and prednisone dosing were correlated with the trough levels

for: PPD1, PPD2, and C1. The tacrolimus-prednisone surfaces indicate synergistic or

antagonistic interactions based upon the respective shape (convex or concave) and the trend

(upward or downward). The color within the plot represents the trough level (ng/ml) according to

the color bar (blue to red).

Fig. S3. PPD tacrolimus-cotrimoxazole interaction plots for patient PPD4. Two-

dimensional (2D) drug interaction maps of tacrolimus and cotrimoxazole dosing correlated with

the trough level for: treatment days 6-12, treatment days 6-15, treatment days 14-23, and

treatment days 14-26. The color within the plot represents the trough level (ng/ml) according to

the color bar (blue to red).

Fig. S4. Retrospective PPD-guided tacrolimus and prednisone dosing optimization for

patients C1 and C2. (A and B) Control patients C1 (A) and C2 (B) PPD-optimized (red) and the

clinically observed (blue) tacrolimus whole blood trough concentration levels (trough levels),

tacrolimus doses, and prednisone doses plotted against dosing days. (C and D) PPD (red) and

clinically observed control (blue) ratios of AUC inside and AUC outside the target range to total

AUC for C1 (C) and C2 (D).

Table S1. Clinical summaries of control patients (C1 to C4) and PPD-guided patients

(PPD1 to PPD4). All patients’ summaries show anonymized demographic information and

treatment parameters.

Rej

ecti

on

None

None

Poss

ible

anti

body

med

iate

d

None

None

Poss

ible

low

gra

de.

No b

iopsy

per

form

ed

None

None

Infe

ctio

n

None

Pneu

mon

ia b

ut

reco

ver

ed

Poss

ible

intr

abdo

min

al

infe

ctio

n,

pre

sum

ed

chola

ngit

is

Bri

ef i

ntr

aven

ous

(IV

) an

tibio

tics

(abx)

for

intr

aabdo

min

al

infe

ctio

n

None

Bil

iary

lea

k -

zosy

n

None

Posi

tive

blo

od

cult

ure

- l

inez

oli

d

Ren

al f

unct

ion

AK

I re

solv

ed

Pre

-ort

ho

topic

liver

tra

nsp

lan

t

(Pre

OL

T)

HD

.

HD

at

dis

char

ge

AK

I re

solv

ed

Acu

te K

idney

Inju

ry (

AK

I)

reso

lved

Pre

OL

T H

D.

HD

at d

isch

arge

AK

I re

solv

ed

AK

I re

solv

ed

Pre

OL

T H

D t

hen

reco

ver

ed

Po

st-o

pera

tiv

e is

sues

No

ne

Res

pir

ato

ry f

ailu

re,

pro

lon

ged

intu

bat

ion

po

st o

per

atio

n,

swal

low

ing

dif

ficu

ltie

s

Per

sist

ent

infe

ctio

n,

po

or

gra

ft

fun

ctio

n,

pla

smap

har

esis

fo

r an

ti-

do

no

r an

tib

od

ies,

bo

rtez

om

ib,

intr

aven

ou

s im

mu

no

glo

bu

lin

(IV

IG),

mu

ltip

le e

xp

lora

tory

lap

rosc

op

ies,

bio

psi

es,

Ttu

be

wit

h

hig

h o

utp

ut

des

pit

e el

evat

ion

,

nee

ded

em

bo

liza

tio

n o

f sp

len

ore

nal

shu

nt,

nee

ded

en

do

sco

pic

ret

rog

rad

e

cho

lan

gio

pan

cre

ato

gra

m a

nd

sten

tin

g o

f d

uct

an

asto

mo

sis/

sten

t

pla

cem

ent

Per

sist

ent

asci

tes

Pro

lon

ged

in

abil

ity

to

sw

allo

w d

ue

to p

rolo

ng

ed

IC

U s

tay

pre

OL

T.

Nee

ded

tu

be

feed

s u

nti

l v

ery

clo

se

to t

ime

of

dis

char

ge

Bil

iary

lea

k

So

me

pro

lon

ged

sw

allo

win

g

dif

ficu

ltie

s b

ut

reco

ver

ed

Per

sist

ent

po

siti

ve

blo

od

cu

ltu

res.

Ev

entu

ally

cam

e o

ff

HD

an

d H

D

lin

e w

as r

emo

ved

Op

erat

ive

issu

es

No

ne

T-t

ub

e p

lace

d.

Sev

ere

isch

emia

rep

erfu

sio

n i

nju

ry,

card

iac

inst

abil

ity

Co

agu

lop

ath

ic,

pac

ked

Ttu

be

pla

ced

.

No

ne

Car

dio

my

op

ath

y,

coag

ulo

pat

hy

,

tem

po

rari

ly c

lose

d.

Tak

en b

ack

fo

r

com

ple

tio

n a

fter

tw

o

day

s

Ro

ux

-en-Y

anas

tom

osi

s.

Mo

rbid

ly o

bes

e

Pre

trea

tmen

t lo

cati

on

ICU

, o

n r

oo

m a

ir,

no

vas

op

ress

ors

,

ence

ph

alo

pat

hic

Inte

nsi

ve

Car

e U

nit

(IC

U),

in

tub

ate

d,

on

hem

od

ialy

sis

(HD

), a

nd

vas

op

ress

ors

Ho

me

Ho

me

ICU

, in

tub

ated

, o

n H

D,

and

vas

op

ress

ors

Ho

me

Ho

me

ICU

, o

n r

oo

m a

ir,

no

vas

op

ress

ors

,

ence

ph

alo

pat

hic

Physi

olo

gic

ME

LD

36

40

13

19

40

25

9

40

Dis

ease

Nonal

coholi

c

Ste

atohep

atit

is

(NA

SH

)

Alc

ohol-

Induce

d

Liv

er D

isea

se

Alc

ohol-

Induce

d

Liv

er D

isea

se,

Hep

atoce

llula

r

Car

cinom

a

Hep

atit

is C

,

Hep

atoce

llula

r

Car

cinom

a

Alc

ohol-

Induce

d

Liv

er D

isea

se

Hep

atit

is C

Hep

atit

is C

,

Hep

atoce

llula

r

Car

cinom

a

NA

SH

Gen

der

F

M

M

M

M

M

M

F

Age

50

39

73

61

53

56

65

54

ID

C1

C2

C3

C4

PP

D1

PP

D2

PP

D3

PP

D4

Table S2. Data used for statistical comparisons in Fig. 5.

Patient

Days post-op in

hospital until

discharge

Days >2 ng/ml

outside of

target range

Ratio of days

>2 ng/ml

outside of

target range

AUC inside

of target

ratio

AUC outside

of target ratio

C1 22 7 0.7 0.058725972 0.941274028

C2 46 2 0.060606061 0.61717296 0.38282704

C3 98 5 0.357142857 0.209903033 0.790096967

C4 29 2 0.111111111 0.589330974 0.410669026

PPD1 33 2 0.095238095 0.629840254 0.370159746

PPD2 30 1 0.090909091 0.671332525 0.328667475

PPD3 22 2 0.2 0.430475768 0.569524232

PPD4 33 1 0.045454545 0.707260047 0.292739953

Table S3. Patient-specific antibiotic and antifungal dosing changes and corresponding

tacrolimus trough levels. A summary is shown of patient-specific correlations between the

trough levels (ng/ml) and the therapeutic co-administration of amikacin, ganciclovir, linezolid,

piperacillin-tazobactam (taz), ciprofloxacin, cotrimoxazole, and fluconazole. (+) are increments

and (-) are decrements of the dosages or the trough levels between treatment period dates (date

vs. date).

Drug Patient Date vs date Change (mg) Trough level ch

ange (ng/ml)

Amikacin (A) and

Ganciclovir (G) PPD1 1/31 vs 2/1 -400 (A)

-110 (G) +1.4

Amikacin (A) and

Linezolid (L) C2 2/7 vs 2/8 -575 (A)

-1200 (L) +3.4

Amikacin (A) and

Piperacillin-Taz (P) C2 2/2 vs 2/3 -150 (A)

-6750 (P) +1.7

Ciprofloxacin C2 1/31 vs 2/1 -400 0

Cotrimoxazole C4 2/4 vs 2/5 -160 -1

C4 2/14 vs 2/16 +160 +0.3

PPD1 2/16 vs 2/17 -160 +0.9

PPD1 2/18 vs 2/19 -160 +0.5

PPD4 2/13 vs 2/14 +320 -4.1

PPD4 2/15 vs 2/16 -320 +1.7

Fluconazole C2 2/18 vs 2/19 -200 -3.4

C4 1/31 vs 2/1 +400 -0.3

PPD4 2/24 vs 2/25 -200 +4.1

Linezolid PPD4 2/23 vs 2/24 -600 +0.7

Table S4. Patient-specific anti-inflammatory and immunosuppressant dosing changes and

corresponding trough levels. A summary is shown of patient-specific correlations of trough

levels (ng/ml) with therapeutic co-administration of methylprednisolone, mycophenolate,

prednisone, and ganciclovir (mg). (+) are increments and (-) are decrements of the dosages or the

trough levels between treatment period dates (date vs. date).

Drug Patient Date vs date Change (mg) Trough level ch

ange (ng/ml)

Methylprednisolone PPD2 2/11 vs 2/12 -120 -1.3

Mycophenolate C1 2/9 vs 2/13 +1000 -7.4

PPD2 2/18 vs 2/23 +500 +2.7

Prednisone C2 2/5 vs 2/6 -2.5 +6.5

PPD1 2/5 vs 2/11 -2.5 -1.5

PPD1 2/14 vs 2/16 -2.5 -0.4

PPD1 2/14 vs 2/18 -2.5 -1.7

Prednisone (P) and

Ganciclovir (G) PPD1 1/30 vs 2/3 -2.5 (P)

-10 (G) -2.9

PPD4 2/17 vs 2/19 -2.5 (P) +125 (G)

-2.6