ORIGINAL ARTICLE

Hepatotoxicity During Maintenance Therapy and Prognosis

in Children With Acute Lymphoblastic Leukemia
Maria S. Ebbesen, MD,* Ulrikka Nygaard, MD, PhD,* Susanne Rosthj, MSc, PhD,w
Ditte Srensen, MSc,w Jacob Nersting, MSc, PhD,*
Kim Vettenranta, MD, PhD,z Finn Wesenberg, MD, DMSc,y
Jon Kristinsson, MD,8 Arja Harila-Saari, MD, PhD,z
and Kjeld Schmiegelow, MD, DMSc*#

Summary: Hepatotoxicity is a known toxicity to treatment of

childhood acute lymphoblastic leukemia. Hepatotoxicity occurs
during maintenance therapy and is caused by metabolites of 6-
Mercaptopurine (6 MP) and Methotrexate (MTX). Our objective
was to investigate the association between alanine amino-
transferases (ALAT) levels and relapse rate. We included 385
patients enrolled in the NOPHO ALL-92 protocol. Data on ALAT
levels, 6 MP and MTX doses, cytotoxic MTX/6 MP metabolites,
and thiopurine methyltransferase (TPMT) activity were pro-
spectively registered. In total, 91% of the patients had a mean
ALAT (mALAT) level above upper normal limit (40 IU/L) and
ALAT levels were positively correlated to 6 MP doses (rs = 0.31;
P < 0.001). In total, 47 patients suered a relapse, no dierence in
mALAT levels were found in these compared with nonrelapse
patients (median, 107 vs. 98 IU/L; P = 0.39). mALAT levels in
patients classied as TPMT high activity (TPMTWT) were higher
than in TPMT low-activity patients (median, 103 vs. 82 IU/L;
P = 0.03). In a Cox regression model risk of relapse was not
associated with ALAT levels (P = 0.56). ALAT levels increased
2.7%/month during the last year of maintenance therapy
(P < 0.001). In conclusion, elevated ALAT levels are associated
with TPMTWT and may indicate treatment adherence in these
patients. If liver function is normal, elevated ALAT levels should
not indicate treatment adaptation.
Key Words: childhood acute lymphoblastic leukemia, hepatotox-
icity, prognosis, maintenance therapy, thiopurines
(J Pediatr Hematol Oncol 2017;00:000000)
M aintenance therapy (MT) with oral low-dose Metho-
trexate (MTX) and oral 6-Mercaptopurine (6 MP) is
a key component in treatment of childhood acute lym-
phoblastic leukemia (ALL). MT is a delicate balance
between ecacy and toxicity.1 Hepatotoxicity is a common
toxicity to MT28 and may result in parenchymal cell injury
and elevated liver enzyme levels.4,5,9 However, hepatotox-
icity is reversible4 and may even be related to a favorable
outcome.6 Despite this, some treatment protocols reduce
drug doses following hepatotoxicity.3,10
To enhance treatment intensity, 6 MP increments are
used. The resulting elevation in methylated metabolites
(MeMP) is associated with hepatotoxicity and a subsequent
rise in serum alanine aminotransferase (ALAT). Thus,
ALAT levels may reect both drug disposition and poten-
tially also adherence to therapy (Fig. 1).
MTX exerts its antineoplastic eect by the cytotoxic
metabolites MTX polyglutamates,11,12 whereas 6 MP does
so by formation of 6-thioguanine nucleotides (TGN)13,14
and MeMP, the latter being catalyzed by thiopurine

Received for publication April 4, 2016; accepted November 30, 2016.

From the *Department of Pediatrics and Adolescent Medicine,
Rigshospitalet; wSection of Biostatistics, Department of Public
Health; #The Institute Clinical Medicine, University of Copenha-
gen, Copenhagen Denmark; zDepartment of Pediatrics, University
Hospital, Tampere, Finland; yDepartment of Pediatrics, The Uni-
versity Hospital, Oslo, Norway; 8Department of Pediatrics, Uni-
versity Hospital, Reykjavik, Iceland; and zDepartment of Womens
and Childrens Health, Karolinska University Hospital and
Karolinska Institutet, Solna, Stockholm, Sweden.
On behalf of the Nordic Society of Pediatric Hematology and Oncology
(NOPHO).
Supported by the Danish Cancer Society (grant no. 9710030), The FIGURE 1. Summarized mechanism of 6-MP/MTX-induced
Danish Childhood Cancer Foundation, The Childrens Cancer hepatotoxicity and myelotoxicity. The 6 MP is converted into
Foundation, Sweden (grant no. 1999/080), The National Medical 6TGN which are incorporated into DNA where they exert mye-
Research Council (grant no. 22-2-0305), The Nordic Cancer Union, lotoxicity. Thiopurine methyltransferase inactivates 6 MP by
The Lundbeck Foundation (grant nos.: 30/98 and 38/99), The Otto formation of methylated metabolites. Together with MTX poly-
Christensen Foundation, The Gangsted Foundation, and The glutamates some of these methylated metabolites inhibit PDNS
Gunnar Jrgensen Foundation. which may enhance incorporation of 6TGN into DNA but also
The authors declare no conict of interest.
cause hepatotoxicity and increase in serum ALAT. ALAT indicates
Reprints: Kjeld Schmiegelow, MD, DMSc, Department of Pediatrics
and Adolescent Medicine, The University Hospital Rigshospitalet, alanine aminotransferase; ANC, absolute neutrofile count;
JMC-4072, Copenhagen, Denmark (e-mail: kschmiegelow@ HGPRT, hypoxanthine-guanine phosphoribosyltransferase; MTX,
regionh.dk). methotrexate; 6 MP, 6-mercaptopurine; PDNS, purine de novo
Copyright r 2017 Wolters Kluwer Health, Inc. All rights reserved. synthesis; TGN, thioguanine nucleotides.

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 Ebbesen et al
methyltransferase (TPMT).15,16 TGN exert their cytotox-
icity through incorporation into DNA (DNA-TG)14 and
MeMP through inhibition of the purine de novo syn-
thesis,17,18 which may enhance DNA-TG incorporation.19,20
One previous study investigated the prognostic sig-
nicance of hepatotoxicity. However, this study was con-
ducted 30 years ago when treatment intensity, the incidence
of elevated ALAT levels, as well as overall survival were
substantially lower than today.6 We hypothesize that
ALAT levels reect both the treatment intensity and
adherence to therapy, and that elevated levels of ALAT are
associated with reduced risk of relapse. We investigated 385
ALL patients with routine ALAT measurements during
MTX/6 MP MT with the aim of improving MTX/6 MP
dose adjustments in case of hepatotoxicity.
MATERIALS AND METHODS
Patients
The Nordic Society of Pediatric Hematology and
Oncology (NOPHO) ALL-92 MT study included 538
patients diagnosed with nonB-cell childhood ALL between
January 1, 1992 and December 31, 1996.21 We excluded 153
patients due to one or more of the following criteria; an
event during MT; allocation to high-risk therapy; Down
syndrome; <3ALAT measurement; no measurement in 1 of
the 2 consecutive maintenance phases (MT phases did or did
not include courses of high-dose MTX (HDM) [see below]).
Thus, 385 patients met the inclusion criteria. The included
patients consisted of 177 girls and 208 boys of whom 207
had standard risk (SR) and 178 intermediate risk (IR) ALL.
Their median age at diagnosis was 4.0 years (range, 1.0 to
14.9 y), and their median white blood cell (WBC) count at
diagnosis was 6.0109/L (50% range, 3.0 to 14.0109/L).
Risk Group Assignment and Treatment
The nonhigh-risk group assignments were based on
age and WBC at diagnosis (SR: age, 2.0 to 9.9 y and
WBC < 10.0 109/L; IR: age, 1.0 to 1.9 or >10.0 y and/or
WBC, 10 to 49.9109/L). Induction and consolidation
therapy have been described previously.2123 MT started at
treatment week 13 (SR) or 32 (IR) and therapy continued
until 2.5 years (SR) or 2 years (IR) after diagnosis. MT
consisted of MTX (20 mg/m2/wk) and 6 MP (75 mg/m2/d).
The NOPHO ALL-92 maintenance study randomized
patients to either; adjusting doses to achieve a WBC
between 1.5 to 3.5 109/L (control group, N = 198 patients)
or adjusting doses based on a combination of WBC and the
product of red blood cell MTX (E-MTX) and TGN (E-
TGN) levels (pharmacology group, N = 187 patients).21
MTX and/or 6 MP dose reductions were only recommended
if ALAT levels were elevated and bilirubin was >50 mmol/L
(ie, 3 times upper normal limit) or coagulation factors II,
VII, and X were <0.50 working units/L.
During the rst year of MT patients received alternate
pulses of: (i) vincristine (2.0 mg/m2 once) and prednisolone
(40 mg/m2/d for 1 wk) and (ii) HDM at 8 intervals (5 g/m2/
24 h with intrathecal. MTX and leucovorin rescue) until 5
courses of HDM had been given.24 These pulses were given
at an interval of 4 weeks.
Blood Samples
E-MTX and E-TGN metabolites were measured in
7529 blood samples a median of 18 and 21 times (SR
patients) or 11 and 12 times (IR patients), respectively.
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J Pediatr Hematol Oncol  Volume 00, Number 00, 2017
E-MTX was analyzed with a radioligand assay (between-
run coecient of variation: 3.0%).25 E-6TGN was deter-
mined with reversed-phase HPLC (between-run coecients
of variation, 8.2%).26,27 Median ALAT measurements
during MT were 25 and 14 times in SR and IR patients,
respectively.
TPMT
TPMT activity was measured in 359 of the 385
patients (between-run coecient of variation: 2.5%).23,28
TPMT activity was determined >8 weeks after the last
blood transfusion. The median TPMT activity was 18.2 IU/
mL (range, 0.6 to 27.6). The antimode of the TPMT activity
distribution was 14 IU/mL.29 Patients carrying 1 allele with
low-activity polymorphisms G460A or A719G or, if TPMT
genotype was not available (n = 381), with TPMT activity
below 14 IU/mL were classied as TPMT low activity
(TPMTLA) (n = 44) and the remaining as TPMT wild-type
(TPMTWT) (n = 315).
Statistical Analysis
For each patient, the mean ALAT (mALAT) during
MT was determined as a weighted average of all registra-
tions, each registration counting until the next registration
or a maximum of 8 weeks (corresponding to a Last
Observation Carried Forward procedure). Weighted mean
absolute neutrole count (mANC) and doses of MTX
(mMTX) and 6 MP (m6MP) were determined in the same
manner.
The Mann Whitney U test was used to compare
groups and Spearman (rs) to explore correlations. MT was
divided into the period including HDM treatment (MT1)
and the period, beginning 8 weeks after last HDM treat-
ment, with only oral 6 MP/MTX treatment (MT2).
To illustrate the dynamics of ALAT levels during MT,
locally weighted scatterplot smoothing (loess regression)
was applied.30
The trend in ALAT levels during MT was analyzed
using a linear mixed model with a random intercept and a
random slope on time in MT for each patient. The ALAT
levels were log transformed due to a right skewed dis-
tribution. The model further included sex, risk group,
randomization group, and TPMT as xed covariates. The
analysis was performed separately for MT1 and MT2.
Event free survival probabilities were determined by
the Kaplan-Meier estimator and the cumulative incidence
of relapse for each risk group was determined using the
Aalen-Johansen estimator accounting for competing risks
(delayed entry at end of MT).
The risk of relapse was analyzed by a Cox regression
model with delayed entry at the end of MT, day 0 being the
date of diagnosis. Patients with competing events were
censored at the timepoint of event. The model was stratied
on risk group and included WBC and age at diagnosis, sex,
randomization group, TPMT, mALAT, mANC, mMTX,
and m6MP. The assumptions of the model were assessed by
graphical examination of the score processes. The mALAT
variable was log2 transformed due to a right skewed dis-
tribution. Patients without TPMT status (n = 26) were not
included in the Cox regression model.
Calculations were performed using SPSS version 22
and R version 3.2.0. P < 0.05 were regarded signicant.
 J Pediatr Hematol Oncol  Volume 00, Number 00, 2017 Hepatotoxicity and Prognosis in Childhood ALL

RESULTS
Hematology and Pharmacology
The median follow-up was 15.5 years (50% range, 13.7
to 16.8 y). In total, 47 patients relapsed (36 involved the
bone-marrow) at a median time of 42 months (range, 28 to
215 mo) from diagnosis, and 8 patients developed a second
cancer. We observed no dierence in mALAT levels in
patients who relapsed compared with patients staying in
remission (107 vs. 98 IU/L, P = 0.39).
Hepatotoxicty During MT
For the entire MT period the median mALAT was
100 IU/L (50% range, 60 to 157 IU/L) and 91% of patients
had a mALAT level above the upper normal limit (40 IU/
L). The TPMTWT patients had higher mALAT levels than
the TPMTLA patients in both MT1 and MT2 (Table 1).
ALAT and 6 MP
A signicant association between m6MP dose and
mALAT levels was observed for both periods of MT
(Table 1), but when stratied by TPMT groups this asso-
ciation was signicant only among TPMTWT patients (the
entire period: TPMTWT; rs = 0.32, P < 0.001 and
TPMTLA; rs = 0.17, P = 0.27).
The mALAT was not associated with age or
randomization. The mALAT was positively correlated to
the number of ALAT measurements from each patient
(median number of measurements = 25). Girls and SR
patients had signicantly higher mALAT levels than boys
and IR patients, respectively. Beyond the association with
risk group and ANC, no change in association between
mALAT and the other variables were observed when
dividing MT in the 2 treatment periods (Table 1).
ALAT Trend During MT
The multiple linear mixed regression analysis for each
MT period showed an increase in ALAT levels of 2.7% per
month during MT2 (P < 0.001). This was not the case in
MT1. Figures 2A, B demonstrate great intrapatient and
interpatient variability in ALAT levels during MT
and Figure 2B shows the increase in ALAT in MT2. The
regression analysis also showed that ALAT levels were
26.7% (P = 0.02) and 19.0% (P = 0.09) higher in
TPMTWT patients compared with TPMTLA patients in
MT1 and MT2, respectively. For patients who stayed in
remission throughout MT there was a signicant correla-
tion between mALAT in MT1, when HDM was also given,
and mALAT in MT2 (rS = 0.53, P < 0.001).
Hepatotoxicity and Relapse Risk
In the Cox regression analysis model describing the
risk of relapse, we investigated whether the eect of (log2)
mALAT was modied by TPMT group. Log mALAT
levels were not signicantly related to relapse risk for
TPMTWT patients (hazard ratio [HR] corresponding to a
doubling of ALAT, 1.09; condence interval [CI], 0.75-1.57;
P = 0.66) or TPMTLA patients (HR, 1.37; CI, 0.47-4.04;
P = 0.57). As these 2 HRs were not signicantly dierent
(P = 0.78), a Cox regression model without interaction
between TPMT group and mALAT was performed which
showed no eect of mALAT on relapse risk (HR, 1.16 for a
doubling of mALAT; CI, 0.70-1.93; P = 0.56) (Table 2).
Very High ALAT Levels
A total of 12 patients had a mALAT level above
350 IU/mL in MT2 and they were all TMPTWT patients.
Their median 6 MP dose was 79 mg/m2/d which was 23%
higher than the median 6 MP dose for the remaining
patients.
DISCUSSION
Two decades ago we published data indicating that
hepatotoxicity during MT was associated with improved
outcome.6 Patients with mALAT above 40 IU/L had a
higher probability of staying in remission compared with

TABLE 1. ALAT in Subgroups

mALAT Entire MT mALAT in MT1* mALAT in MT2w
Median mALAT P Median mALAT P Median mALAT P
Total 99.5 80.7 109.3
Male/female 90.9/110.9 0.01z 75.8/87.6 0.03 99.1/122.2 0.04
TPMTLA/TPMTWTy 82.4/102.9 0.03z 54.4/84.9 0.03 84.3/115.4 0.02
SR/IR treatment 108.9/88.4 0.003z 85.0/75.0 0.24 122.2/97.3 0.003
Control/pharmacology8 97.1/103.9 0.30z 77.3/82.6 0.39 100.9/115.8 0.17
rs rs rs
Age (y) 0.057 0.27 0.090 0.08 0.005 0.92
No. ALAT-samples 0.373 < 0.001 0.401 < 0.001 0.28 < 0.001
m6MP dose 0.307 < 0.001 0.289 < 0.001 0.273 < 0.001
mMTX dose 0.18 < 0.001 0.19 < 0.001 0.125 0.01
mE-TGN  0.103 0.04  0.126 0.01  0.109 0.03
mE-MTX 0.072 0.15 0.071 0.17 0.072 0.17
mANC 0.127 0.01 0.025 0.62 0.103 0.05
*Period of MT including high-dose MTX.
wPeriod of MT starting 8 weeks after last high-dose MTX treatment with only oral 6 MP/MTX treatment.
zThe Mann Whitney U test.
yTPMT low-activity group/TPMT wild-type group (dened in text).
8Pharmacology group = dose adjustments by red blood cell levels of MTX and 6 MP metabolites. Control group.20
ALAT indicates alanine aminotransferase; ANC, absolute neutrole count; IR, intermediate risk; MT, maintenance therapy; MTX, methotrexate;
mE-MTX, product of red blood cell MTX;6 MP, 6-mercaptopurine; rs, The Spearman Rank correlation; SR, standard risk; TGN, thioguanine nucleotides;
mE-TGN, product of red blood cell TGN; TPMT, thiopurine methyltransferase.

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 Ebbesen et al J Pediatr Hematol Oncol  Volume 00, Number 00, 2017

FIGURE 2. ALAT dynamic in ALL patients during MT. Grey lines demonstrate 7 randomly selected patients ALAT levels during MT1*
and MT2w, respectively, and shows great interpatient and intrapatient variability in ALAT levels. The black Loess line depicts the trend in
the ALAT levels in MT1 and MT2, respectively, and shows a small increase in ALAT during MT2. *Period of MT including high-dose MTX.
wPeriod of MT starting 8 weeks after last high-dose MTX treatment with only oral 6 MP/MTX treatment. ALAT indicates alanine
aminotransferase; ALL, acute lymphoblastic leukemia; MT, maintenance therapy; MTX, methotrexate.

patients with ALAT levels below 40 IU/L (0.83 vs. 0.63,
P = 0.03). However, those ndings may be less relevant for
relapse with contemporary ALL protocols due to increased
treatment intensity and better adherence to therapy today.
Thus, the present study demonstrated far higher ALAT
levels than that previous report and no correlation of
ALAT to relapse risk.
The observed association between a rise in ALAT
levels and 6 MP dose must be interpreted with caution due
to the possibility of physicians having decreased 6 MP dose
due to high ALAT levels. However, it is worth noticing the
positive association between ALAT levels and 6 MP doses.
A previous study in the same patient cohort found a sig-
nicant association between 6 MP dose and increased
ALAT levels following HDM administration.31 The
mechanism of MTX and MeMP induced hepatotoxicity is
unknown. However, both metabolites inhibit purine de
novo synthesis and MeMP may be toxic to hepatic cells
through this inhibition.17 Furthermore, previous studies
demonstrated association between ALAT and MeMP lev-
els.32,33 Thus, low ALAT levels may in part reect poor
adherence or reduced bioavailability of 6 MP, at least for
TPMTWT patients. Inferior treatment adherence is asso-
ciated with higher risk of relapse,3439 and may be con-
rmed through measurements of blood levels of 6 MP and
MTX metabolites, but these are only routinely available in
a few countries. Importantly, the very few patients with
consistently low ALAT levels, and the lack of association
between ALAT and relapse rate we found in this study may
indicate improved treatment adherence today.
Our ndings extend results from our earlier study23 in
which a signicantly higher relapse rate was found for
patients classied as TPMTWT compared with TPMTLA
patients across ALL subtypes.
It is important to consider the long-term side eects for
patients having high ALAT levels during MT. Previous
studies showed that high ALAT levels normalize rapidly after
discontinuation of therapy,24,7,32 but studies from the 1970s
and 1980s demonstrated high incidence of hepatic brosis
after therapy for childhood ALL.5,9,40 Parker et al inves-
tigated liver biopsies due to clinical abnormalities and/or
elevated serum enzymes in 8 of 36 children receiving oral MT.
One of the patients had micronodular cirrhosis and 3 had
brous tissue in portal tracts.5 Harb et al9 found brosis in all
liver biopsis from 11 consecutive children treated with MTX
and 6 MP. The high risk of hepatic brosis could reect the
high prevalence of hepatotropic viruses at that time due to
unscreened blood transfusions. However, the most recent
study evaluating liver histology following ALL therapy
according to the NOPHO ALL-92 protocol, without coinci-
dence of hepatotropic viruses, found mainly microvesicular
fatty inltration and siderosis. Only 3 of 27 patients had mild
hepatic brosis.4 Symptomatic fasting hypoglycemia has been
found as a complication in ALL children receiving 6 MP4143
and has been associated with high levels of MeMP.44 Shifting
to morning or twice a day dosage in attempt to reduce

TABLE 2. Cox Multivariable Regression Analysis of Risk of Relapse

Hazard Ratio 2.5% 97.5% P
WBC* 1.00 0.96 1.03 0.86
Age (y) 1.01 0.91 1.11 0.88
Pharmacology group. 2.08 1.11 3.89 0.02
Malew 2.07 1.05 4.07 0.04
mMTX dose 1.16 1.04 1.28 0.005
m6MP dose 0.99 0.96 1.01 0.34
mANC 2.10 1.19 3.68 0.01
TPMTWT 2.21 0.67 7.33 0.20
mALAT 1.16w 0.70 1.93 0.56
*White blood cell count at diagnosis.
wCorresponding to a doubling of mALAT.
mALAT indicates mean alanine aminotransferase; mANC, mean absolute neutrole count; m6MP, mean 6-mercaptopurine; mMTX, mean methotrexate;
WBC, white blood cell; TPMT, thiopurine methyltransferase.

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 J Pediatr Hematol Oncol  Volume 00, Number 00, 2017
hypoglycemia resulted in decreased MeMP levels but the
impact on relapse risk is unknown.44
In conclusion, persistently elevated ALAT during MT 18.
in part indicate treatment adherence and is likely to be
associated with TPMTWT. Elevated ALAT should not in
itself indicate treatment adaptation if liver function is 19.
normal.
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