Original Reports | Pediatric Oncology

Four Additional Doses of PEG-L-Asparaginase During the

Consolidation Phase in the AIEOP-BFM ALL 2009 Protocol Do
Not Improve Outcome and Increase Toxicity in High-Risk ALL:
Results of a Randomized Study
Valentino Conter, MD1 ; Maria Grazia Valsecchi, PhD2,3 ; Gunnar Cario, MD4; Martin Zimmermann, PhD5 ; Andishe Attarbaschi, MD6,7 ;
Jan Stary, MD8 ; Felix Niggli, MD9 ; Luciano Dalla Pozza, MD10; Sarah Elitzur, MD11 ; Daniela Silvestri, MSc1; Franco Locatelli, MD12 ;
Anja Möricke, MD4; Gernot Engstler, MD6; Petr Smisek, MD8 ; Nicole Bodmer, MD9; Draga Barbaric, MD13; Shai Izraeli, MD11 ;
Carmelo Rizzari, MD2,14 ; Joachim Boos, MD15; Barbara Buldini, MD16 ; Massimo Zucchetti, PhD17 ; Arend von Stackelberg, MD18;
Cristina Matteo, PhD17 ; Thomas Lehrnbecher, MD19 ; Claudia Lanvers-Kaminsky, PhD15; Giovanni Cazzaniga, PhD1,2 ; Bernd Gruhn, MD20;
Andrea Biondi, MD2,14 ; and Martin Schrappe, MD4

DOI https://doi.org/10.1200/JCO.23.01388

ABSTRACT ACCOMPANYING CONTENT

PURPOSE The AIEOP-BFM ALL 2009 protocol included, at the end of the induction phase, a Appendix
randomized study of patients with high-risk (HR) ALL to investigate if an in- Protocol
tensive exposure to pegylated L-asparaginase (PEG-ASNASE, 2,500 IU/sqm once
a week 3 4) on top of BFM consolidation phase IB allowed us to decrease minimal Accepted September 26, 2023
residual disease (MRD) and improve outcome. Published December 14, 2023
PATIENTS AND A total of 1,097 patients presented, from June 2010 to February 2017, with one or
METHODS more of the following HR criteria: KMT2A::AFF1 rearrangement, hypodiploidy,
prednisone poor response, poor bone marrow response at day 15 (Flow J Clin Oncol 00:1-12
MRD ≥10%), or no complete remission (CR) at the end of induction. Of them, © 2023 by American Society of
809 (85.1%) were randomly assigned to receive (404) or not receive (405) four Clinical Oncology
weekly doses of PEG-ASNASE.
RESULTS By intention to treat (ITT) analysis, there was no significant difference in the
proportion of patients with polimerase chain reaction MRD ≥5 3 1024 at the View Online
Article
end of phase IB in the experimental versus control arm (13.9% v 17.0%,
P 5 .25). The 5-year event-free survival (median follow-up 6.3 years) by
ITT in the experimental and control arms was 70.4% (2.3) versus 75.0% (2.2;
P 5 .18), and the 5-year overall survival was 81.5% (2.0) versus 84.0% (1.9;
P 5 .25), respectively. The corresponding 5-year cumulative incidence of
death in CR was 9.5% (1.5) versus 5.7% (1.2; P 5 .08), and that of relapse was
17.7% (1.9) versus 17.2% (1.9), respectively (P 5 .94). Adverse reactions in
phase IB occurred in 22.2% and 8.9% of patients in the experimental and
control arm, respectively (P < .001).
CONCLUSION Additional PEG-ASNASE in phase IB did not translate into a benefit for de-
creasing relapse incidence but was associated with higher toxicity. Further
improvements with conventional chemotherapy might be difficult in the
context of intensive treatment protocols.

INTRODUCTION of Consolidation phase IB have poor outcome despite

administration of subsequent intensive chemotherapy
Improving the outcome of children and adolescents with blocks and allogeneic hematopoietic cell transplantation
ALL presenting with unfavorable features remains a major (allo-HCT).1-4 These findings raised the question as to
challenge. Results of the AIEOP-BFM (Associazione Italiana whether earlier treatment interventions might reduce
di Ematologia e Oncologia Pediatrica - Berlin Frankfurt MRD levels and the risk of relapse and improve outcome.
Muenster) ALL 2000 study showed that patients with high The addition of L-asparaginase to consolidation or sub-
levels of minimal residual disease (MRD ≥5 3 1024) at the end sequent treatment phases has been used successfully in

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 Conter et al

CONTEXT

Key Objective
Does additional L-asparaginase in the consolidation phase improve minimal residual disease (MRD) response and outcome
in children with high-risk ALL (HR-ALL)?

Knowledge Generated
In the AIEOP-BFM ALL 2009 study, 809 patients with HR-ALL were randomly assigned at the end of induction to receive
weekly PEG-L-asparaginase (2500 IU/sqm once a week 3 4) on top of consolidation phase IB. Compared with the control
arm, in the experimental arm there were a modest decrease in MRD, similar relapse rate, and increased toxicity.

Relevance (S. Bhatia)

These findings suggest the need for innovative therapeutic approaches in high-risk ALL patients that would reduce relapse
risk and not increase the toxicity profile.*

*Relevance section written by JCO Associate Editor, Smita Bhatia, MD, MPH, FASCO.

many protocols with improvement of outcomes in childhood end of Consolidation phase IB (day 178) who, by definition,
ALL.5-10 On the basis of these considerations, the AIEOP-BFM are not part of this report. The AIEOP-BFM ALL 2009 study
ALL 2009 protocol was designed to include a randomized was approved by the ethical committees of all participating
treatment question early after induction therapy for patients institutions and was conducted in accordance with the
with high-risk (HR) features. Declaration of Helsinki. Patients entered the study only
after informed consent was obtained from parents or legal
The aim was to investigate if the addition of intensive exposure guardians.
to high-dose pegylated L-asparaginase (PEG-ASNASE) to BFM
Consolidation phase IB could reduce MRD levels (primary end Diagnostic Studies
point) and improve outcome. Here, we report the results of this
randomized study. The diagnosis of ALL was confirmed using cytomorphology
when ≥25% of lymphoblastic cells were present in the BM.
PATIENTS AND METHODS For all samples, FCM immunophenotyping11 and screening
for ETV6::RUNX1, BCR::ABL1, and KMT2A::AFF1 fusion tran-
Patients scripts and chromosome numbers (or DNA Index) were
performed. CR was defined as a regenerating BM with <5% of
From June 1, 2010, to February 28, 2017, a total of 6,136 leukemia blast cells and no evidence of extramedullary in-
patients with Philadelphia chromosome–negative ALL, age 1- volvement. MRD analysis procedures by FCM or PCR have
17 years, were enrolled in the AIEOP-BFM ALL 2009 study been reported elsewhere.1,2,11-13
(EudraCT number 2007-004270-43). Participating study
centers were in Australia/New Zealand, Austria, Czech Treatment Protocol and Random Assignment in
Republic, Germany, Israel, Italy, and Switzerland. Of these HR Patients
patients, 1,097 were stratified as HR at the end of
Induction phase IA (day 33), with at least one of the fol- Treatment details of the AIEOP-BFM ALL 2009 study have
lowing findings: KMT2A::AFF1 rearrangement, hypodip- been recently reported.14 Briefly, after 7 days of prephase
loidy (<45 chromosomes or DNA Index <0.8), prednisone with steroid therapy (prednisone) and one dose of ITMTX, all
poor response (PPR; patients with at least 1,000 blast patients received induction protocol IA, which included
cells/mL in peripheral blood after 7 days of prednisone prednisone (60 mg/sqm daily in three divided doses for
(PDN) monotherapy and one injection of intrathecal 3 weeks plus tapering doses over 9 days) or dexamethasone
methotrexate [ITMTX]), high levels of residual leukemia (10 mg/sqm daily in three divided doses for 3 weeks plus
on day 15 of induction therapy (≥10% of blast cells in the tapering doses over 9 days in patients with T-ALL with
bone marrow (BM) by flow cytometry [FCM]), or failure to prednisone good response [PGR]), vincristine (1.5 mg/sqm,
achieve morphological or clinical complete remission (CR; maximum 2 mg, once a week for 4 weeks), daunorubicin
≥5% blasts in BM or persistence of extramedullary disease) at (30 mg/sqm, once a week for 2 or 4 weeks), one dose of
the end of Induction phase IA (Fig 1). The final HR group also PEG-ASNASE (2,500 IU/sqm) at day 12 and at day 26,
included patients defined as HR based solely on polimerase and two (in CNS1) or four doses (in CNS2 and CNS3) of
chain reaction minimal residual disease (PCR-MRD) at the ITMTX. Patients with T-ALL and PPR received one dose of

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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

Total enrollment (N = 6,136) Final without HR (n = 4,729)

Final HR (n = 1,407)

HR at the end of inductiona Not eligible for random assignment (n = 146)

(n = 1,097) Discontinuation or substantial change (n = 10)
of protocol therapy by start of phase IB
Deaths before random assignment (n = 6)
PEG-ASNASE allergy (n = 19)
Pancreatitis (n = 7)
Thrombosis (n = 31)
Other toxicities preventing random at (n = 73)
the physician’s discretion

Eligible for random assignment

Not randomly assigned (n = 142)
(n = 951)
Patients'/guardians' refusal (n = 112)
Other reasons (n = 30)

Randomly assigned (n = 809)

Experimental treatment with four Control arm with 0 doses of ITT

doses of PEG-ASNASE (n = 404) PEG-ASNASE (n = 405) analysis

Received experimental treatment (n = 362) Received standard treatment (n = 403)

Four doses (n = 307) Misclassified as HR (n = 1)
Three doses (n = 17) Shifted to the experimental arm (n = 1)
Two doses (n = 17) Four doses (n = 1)
One dose (n = 21)
Deaths before treatment start (n = 7)
Misclassified as HR (n = 1)
Shifted to the control arm (n = 34)
Patients’/guardians’ refusal (n = 8)
Physician’s decision (n = 18)
Other reasons (n = 8)

With at least one dose (n = 363) No doses (n = 437) As treated

FIG 1. CONSORT diagram. aIt includes two patients who were misclassified as HR at the end of
induction and randomly assigned (not included in 1,407 final HR). HR, high-risk; PEG-ASNASE,
pegylated L-asparaginase.

cyclophosphamide (1 g/sqm) at day 10. Patients with B-cell drugs (with one dose of ITMTX in each block for all patients
precursor (BCP) ALL and no HR criteria (at day 115), who and one additional dose in block 2 in CNS3 patients). Re-
were ETV6::RUNX1-positive, or with <0.1% of blast cells at sistance was defined as failure to achieve CR by the end of
day 15 BM by FCM were eligible to be randomly assigned to the third HR block. In patients who did not go onto allo-
receive two versus four doses of daunorubicin. HCT, chemotherapy was continued with three Reinduction
phases (Protocol III 33, each with ITMTX 32 in CNS1 and
Patients classified as HR at the end of Induction phase IA were two patients, 33 in CNS3 patients) and maintenance
eligible to be randomly assigned to receive PEG-ASNASE therapy with oral 6-MP and MTX for a total duration of
(2,500 IU/sqm once a week for 4 weeks, supplied during the 24 months from diagnosis. Preventive cranial radiotherapy
study period in chronological order by companies medac (CRT, 12 Gy) was given after first Protocol III to HR patients
GmBH, Sigma-Tau, and Baxalta) on top of Consolidation 2 years and older who had T-ALL and those who were HR
phase IB, which included 6-MP (one dose, 60 mg/sqm because of hypodiploidy, or positive for KMT2A::AFF1, or did not
daily for 28 days), cyclophosphamide (a single dose of 1 g/ achieve CR at the end of phase IA, or had high levels of MRD
sqm on day 1 and day 29), cytosine arabinoside (75 mg/sqm, after the Consolidation phase. Therapeutic CRT was given to
one dose daily 4 days a week for 4 weeks), and ITMTX (on CNS3 patients (12 Gy if younger than 2 years, 18 Gy if older). All
days 10 and 24; Appendix FigA 1B [online only]). Thereafter, other HR patients received protracted ITMTX (36) during
HR patients received three blocks of non–cross-resistant maintenance. Overall, the treatment schedule for HR patients

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 Conter et al

(not receiving allo-HCT) included 8 or 12 doses of PEG- A sample size of 562 patients was originally planned to detect a
ASNASE, depending on the random assignment in phase IB reduction in the rate of MRD highly positive patients from 33%
(Appendix Fig A1). Allo-HCT was indicated after the third HR to 22% at TP2 (80% power, two-sided test with 5% type I
Block for patients with KMT2A::AFF1 rearrangement or hypo- error). A Data and Safety Monitoring Committee periodically
diploidy (<44 chromosomes or DNA index <0.8) and positive supervised the study progress and approved the prolongation
MRD (or absence of MRD data) at the end of Induction phase IA, of the study enrollment beyond June 2016 because of a
or no CR at the end of phase IA or high MRD level at day 78 (≥5 3 reassessed sample size accounting for a different baseline
1024), or T-ALL PPR without MRD data at day 78. observed (19% instead of 33% of MRD highly positive pa-
tients): 910 randomly assigned patients were considered ap-
End Points and Statistical Analysis propriate to detect a reduction in MRD at TP2 from 19% to 12%
(control v experimental arm, 80% power, two-sided test with
The primary end point in this study was the rate of PCR-MRD a 5% type I error). At study closure, the sample size was 809,
highly positive (≥5 3 1024) patients at TP2 (timepoint 2, end of giving a 75% power, with the other conditions being the same.
Consolidation phase IB). The main secondary end point was
event-free survival (EFS), defined as the time from random Random assignment was performed by day 140 by
assignment to the date of last follow-up (censored) or first computer-generated random allocation and stratified by
event. Events were resistance to treatment, relapse, death, or country in accordance with blocks of four patients.
second malignant neoplasm, whichever occurred first. Overall
survival (OS) was defined as the time from random assign- Patients’ follow-up was updated at June 2021. The median
ment to death from any cause or last follow-up (censored). follow-up time was 6.3 years from random assignment.
EFS and OS were estimated according to the Kaplan-Meier
method with Greenwood SE, indicated in parentheses, and RESULTS
compared according to the log-rank test. Cumulative inci-
dence curves for relapse or death were estimated adjusting for Patients and Random Assignment
competing risks and compared with the Gray test. Analyses
were performed using SAS 9.4. The cohort of 6,136 patients had an overall 5-year EFS and
OS of 84.3% (0.5) and 92.3% (0.4; Fig 2A). Of these, according
Primary and secondary end points were analyzed primarily to stratification criteria, 1,407 (22.9%) were finally classified
by the assigned treatment arm (intention to treat [ITT]) as HR and had a 5-year EFS and OS of 70.6% (1.3) and 81.5%
and in addition with an as-treated approach (patients (1.1; Fig 2B).
treated with at least one dose of PEG-ASNASE included in
the experimental arm, and in the control arm otherwise). As shown in Figure 1, 1,097 patients were classified as HR
The conditional logistic and the Cox regression model were according to criteria assessed by the end of Induction phase
applied to analyze end points, adjusting for relevant IA (day 33) and are the patients of interest for random as-
characteristics (sex, age, immunophenotype, WBC count at signment for additional PEG-ASNASE in consolidation. Of
diagnosis, and early response indicators). these, 146 were not eligible to the randomly assigned study

A 6,136 Patients
B 1,407 Patients
1.0 1.0

0.8 0.8
Probability

Probability

0.6 0.6

0.4 0.4

5-year probability 5-year probability

0.2 0.2
Survival 482 deaths 92.3% (0.4) Survival 266 deaths 81.5% (1.1)
EFS 977 events 84.3% (0.5) EFS 417 events 70.6% (1.3)

0 1 2 3 4 5 0 1 2 3 4 5

Time Since Diagnosis (years) Time Since Diagnosis (years)

Survival 6,136 5,867 5,698 5,324 4,940 3,939 Survival 1,407 1,283 1,209 1,104 1,009 804
EFS 6,136 5,813 5,532 5,030 4,561 3,602 EFS 1,407 1,250 1,142 998 891 704

FIG 2. Overall 5-year EFS and overall survival of (A) the entire cohort of 6,136 and (B) the subgroup of HR patients according to final risk
classification. EFS, event-free survival; HR, high-risk.

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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

TABLE 1. Characteristics of Randomly Assigned Patients by Assigned Treatment Arm (ITT population)

Experimental Arm Control Arm

Characteristic With PEG-ASNASE Without PEG-ASNASE Total

Total, No. 404 405 809
Sex, No. (%)
Male 259 (64.1) 258 (63.7) 517 (63.9)
Female 145 (35.9) 147 (36.3) 292 (36.1)
Age, years, No. (%)
1-5 164 (40.6) 151 (37.3) 315 (38.9)
6-9 85 (21.0) 100 (24.7) 185 (22.9)
10-14 102 (25.3) 106 (26.2) 208 (25.7)
15-17 53 (13.1) 48 (11.8) 101 (12.5)
WBC, 3109/L, No. (%)
<20 164 (40.6) 172 (42.5) 336 (41.5)
20-100 113 (28.0) 115 (28.4) 228 (28.2)
≥100 127 (31.4) 118 (29.1) 245 (30.3)
CNS involvement, No. (%)
CNS1/2 364 (97.3) 360 (96.3) 724 (96.8)
CNS3 10 (2.7) 14 (3.7) 24 (3.2)
Not known 30 31 61
Immunophenotype, No. (%)
BCP-ALL 257 (64.1) 260 (64.8) 517 (63.9)
T ALL 144 (35.9) 141 (35.2) 285 (35.2)
Other/not known 3 4 7 (0.9)
ETV6::RUNX1, No. (%)
Positive 21 (5.2) 25 (6.2) 46 (5.7)
Negative 382 (94.8) 378 (93.8) 760 (95.3)
Not known 1 2 3
KMT2A::AFF1, No. (%)
Positive 16 (4.0) 9 (2.2) 25 (3.1)
Negative 388 (96.0) 394 (97.8) 782 (96.9)
Not known 0 2 2
Hypodiploidy <45 chromosomes, No. (%)
Positive 20 (5.0) 23 (5.7) 43 (5.3)
Negative 382 (95.0) 379 (94.3) 861 (94.7)
Not known 2 3 5
PDN response, No. (%)
Good 191 (47.9) 193 (48.3) 384 (48.1)
Poor 208 (52.1) 207 (51.7) 415 (51.9)
Not known 5 5 10
FCM-MRD day 115, No. (%)
<0.1% 24 (6.1) 11 (2.8) 35 (4.5)
0.1%-10% 90 (22.9) 86 (22.0) 176 (22.4)
≥10% 279 (71.0) 294 (75.2) 573 (73.1)
Not known 11 14 25
Response to Ia, No. (%)
CR 351 (88.9) 373 (92.8) 724 (90.8)
No CR 44 (11.1) 29 (7.2) 73 (9.2)
Not known/not applicable 9 3 12
(continued on following page)

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 Conter et al

TABLE 1. Characteristics of Randomly Assigned Patients by Assigned Treatment Arm (ITT population) (continued)

Experimental Arm Control Arm

Characteristic With PEG-ASNASE Without PEG-ASNASE Total

PCR-MRD day 133, No. (%)
Negative 32 (8.6) 42 (11.3) 74 (10.0)
Positive <5 3 10 24
101 (27.3) 110 (29.6) 211 (28.4)
Positive ≥5 3 1024 237 (64.1) 220 (59.1) 457 (61.6)
Not performed 34 33 67

Abbreviations: BCP-ALL, B-cell precursor-ALL; CR, complete remission; FCM-MRD, flow cytometry-minimal residual disease; ITT, intention to treat;
PDN, prednisone; PCR-MRD, polimerase chain reaction minimal residual disease; PEG-ASNASE, pegylated L-asparaginase; T-ALL, T acute
lymphoblastic leukemia.

(see Fig 1 for details). Of the remaining 951 eligible patients, experimental treatment, 34 did not receive PEG-ASNASE
809 (85.1%) were randomly assigned to receive (n 5 404) or (shifted to the control arm); of those (n 5 362) treated with
not receive (n 5 405) four weekly doses of PEG-ASNASE PEG-ASNASE, 307 (84.8%) received all the planned four
during Consolidation phase IB. doses. Reasons for the reduced number of doses were allergic
reaction to PEG-ASNASE (n 5 15), pancreatitis (n 5 6),
Features of randomly assigned patients are shown in Table 1. thrombosis (n 5 5), death (n 5 1), other medical reasons
Overall, 38.2% of patients were adolescents (10 years and (n 5 22), and other nonmedical reasons (n 5 6). In the control
older at diagnosis), 30.3% had hyperleukocytosis at diagnosis arm (standard treatment), one patient received the experi-
(WBC count ≥100 3 109/L), 35.2% had T-ALL, and 3.1% had mental treatment and one additional patient was considered
KMT2A::AFF1 rearrangement. By early response to treatment, retrospectively not eligible to HR. Thus, the as-treated
51.9% had PPR and 73.1% had high levels of FCM MRD (≥10% population includes 363 patients who received at least
blasts in the BM) at day 15; 9.2% were not in morphological one dose of PEG-ASNASE during phase IB and 437 patients
remission, and 61.6% had high PCR MRD (≥5 3 1024) at the who did not (Fig 1).
end of Induction phase IA. Prognostic features by assigned
arm were well balanced, with a slightly higher frequency of
patients with no CR at the end of phase IA or KMT2A::AFF1 Primary Outcome Analysis—ITT Population
rearrangement in the experimental arm and of patients with
higher FCM MRD levels at day 15 in the control arm. Table 2 presents the results on the primary end point in 732
patients with available MRD data among 809 randomly
Random assignment was performed at a median time of 31 days assigned patients (90.5%). The proportion of patients with
from diagnosis (IQR, 27-36 days). As shown in Figure 1, seven PCR MRD ≥5 3 1024 at the end of Consolidation phase IB in
patients randomly assigned to receive PEG-ASNASE died the experimental versus control arm was 13.9% versus 17.0%
before the planned start of randomized treatment and one with no significant difference (P 5 .25). A logistic model
additional patient was considered retrospectively not eli- confirms this finding (odds ratio, 0.77 [95% CI, 0.51 to 1.17],
gible to HR. Among 404 patients randomly assigned to the P 5 .22; Appendix Table A1, model A). The proportion was

TABLE 2. Primary End Point on the Basis of PCR-MRD at the End of Consolidation Phase IB (day 178, TP2) in 732 Randomly Assigned Patients
With Available Data by Assigned Treatment Arm (ITT population) and by Immunophenotype Subgroup (P values refer to the comparison of the
proportions of patients with MRD POS ≥5 3 1024 in the two arms)

Total BCP-ALL T ALL

With Without With Without With Without

PCR-MRD at Day 178 PEG-ASNASE PEG-ASNASE PEG-ASNASE PEG-ASNASE PEG-ASNASE PEG-ASNASE
No. of patients 367a 365b 241 246 124 116
Negative or positive NQ, 267 (72.7) 260 (71.2) 186 (78.2) 186 (75.6) 79 (63.7) 72 (62.0)
No. (%)
Positive <5 3 1024, No. (%) 49 (13.4) 43 (11.8) 27 (11.3) 21 (8.5) 22 (17.7) 22 (19.0)
Positive ≥5 3 10 , No. (%)
24
51 (13.9) 62 (17.0) 28 (11.6) 39 (15.9) 23 (18.6) 22 (19.0)
P .25 .17 .93

Abbreviations: BCP-ALL, B-cell precursor-ALL; MRD, minimal residual disease; NQ, not quantifiable; PCR-MRD, polimerase chain reaction minimal
residual disease; PEG-ASNASE, pegylated L-asparaginase.
a
Thirty-seven patients did not have the MRD level measured at day 178 (14 deaths, 23 lack of markers).
b
Forty patients did not have the MRD level measured at day 178 (1 death, 39 lack of markers).

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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

TABLE 3. Events After Random Assignment (secondary end point) by Assigned Treatment Arm (ITT population)

Experimental Arm Control Arm

Status With PEG-ASNASE Without PEG-ASNASE Total

Randomly assigned patients, No. 404 405 809
Resistant by protocol, No. (%) 2 (0.5) 1 (0.3) 3 (0.4)
Deaths, No. (%) 38 (9.4) 25 (6.2) 63 (7.8)
Before the start of randomized treatmenta 7 (1.7) 0 7 (0.9)
Phase IBb 7 (1.7) 1 (0.2) 8 (1.0)
After phase IB (CHEMO)c 14 (3.5) 12 (3.0) 26 (3.2)
After allo-HCT 10 (2.5) 12 (3.0) 22 (2.7)
Relapses, No. (%) 72 (17.8) 72 (17.8) 144 (17.8)
BM isolated 41 (10.2) 47 (11.6) 88 (10.9)
BM combined 14 (3.5) 12 (3.0) 26 (3.2)
CNS isolated 12 (3.0) 8 (2.0) 20 (2.5)
TESTIS isolated 1 (0.2) 2 (0.5) 3 (0.4)
Other 4 (1.0) 3 (0.7) 7 (0.8)
Second malignant neoplasm, No. (%) 8 (2.0) 8 (2.0) 16 (2.0)
Alive in CCR, No. (%) 284 (70.3) 299 (73.8) 583 (72.0)

Experimental Arm Control Arm

HCT Status With PEG-ASNASE Without PEG-ASNASE Total

Allo-HCT eligibility, No. (%) 108 (26.7) 105 (25.9) 213 (26.3)
Allo-HCT performed, No. (%) 87 (21.5) 90 (22.2) 177 (21.9)

Abbreviations: allo-HCT, allogeneic-hematopoietic cell transplantation; BM, bone marrow; CCR, continuous complete remission; PEG-ASNASE,
pegylated L-asparaginase.
a
Cause of death for all seven patients was infection.
b
Cause of death for the seven patients who were assigned to the experimental arm was infection (n 5 5, of which one in a patient who shifted to the
control arm), pancreatitis (n 5 1), and thrombosis (n 5 1). In the control arm, death was due to hemophagocytic lymphohistiocytosis (which started
in phase IA and aggravated in phase IB).
c
Causes of deaths were infection in 21, hemophagocytic lymphohistiocytosis in one, sinusoidal obstruction syndrome in one, GI complications in
one, and not known in two.

11.6% versus 15.9% (P 5 .17) in BCP-ALL and 18.6% versus in CR after phase IB were 24 versus 24 in the experimental
19.0% in T-ALL (P 5 .93). We also explored the effect of versus control arm (10 v 12 after allo-HCT in CR1, per-
treatment intensification on MRD clearance in the sub- formed in 87 and 90 patients, respectively). Three patients
group of 453 patients (236 in the experimental arm and were resistant to treatment (2 v 1); 144 relapsed (72 v 72),
217 in the control arm) who had high MRD (≥5 3 10 24 ) at with no relevant difference by site; and 16 had a second
the end of phase IA. At TP2, MRD was still high in 21.2% (n 5 malignant neoplasm (8 v 8). See Table 3 for details and
50) and 27.7% (n 5 60) of patients in the experimental and causes of death.
control arm, respectively (P 5 .11; Appendix Table A2), again
with a more marked difference in the BCP-ALL (20.8% in 130 v The overall 5-year EFS by ITT was 70.4% (2.3) and 75.0%
29.9% in 127 patients), as compared with T acute lympho- (2.2) in the experimental and control arm (P 5 .18, Fig 3A),
blastic leukemia (T-ALL) (21.9% in 105 v 24.1% in 87 patients). respectively, and was 71.6% (2.5) and 75.3% (2.5) by cen-
soring allo-HCT in first CR (P 5 .23). OS figures were 81.5%
Secondary Outcome Analysis (EFS)—ITT Population (2.0) and 84.0% (1.9; P 5 .25, Fig 3B). The cumulative inci-
dence of relapse (CIR) at 5 years was 17.7% (1.9) versus 17.2%
By ITT analysis, there were more toxic deaths during Con- (1.9) in the experimental versus control arm (Fig 3C). The
solidation phase IB among patients randomly assigned to small no significant difference in EFS is explained by the risk
PEG-ASNASE versus controls: seven of them, all in the of death as the first event (9.5 [1.5] v 5.7 [1.2], Figure 3D).
experimental arm, occurred before starting randomized When a Cox model was applied to adjust the estimate of the
treatment; eight additional deaths (7 v 1) occurred in phase treatment effect on EFS (by ITT analysis), the hazard ratio was
IB after the time of experimental treatment start. Deaths 1.05 (95% CI, 0.78 to 1.42; P 5 .73), indicating no significant

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 Conter et al

A B
809 Patients 809 Patients
1.0 1.0

0.8 0.8
EFS (probability)

OS (probability)
0.6 0.6

0.4 0.4
No. of No. of No. of No. of
Patients Events 5-year EFS Patients Deaths 5-year OS
0.2 Yes 404 120 70.4% (2.3) 0.2 Yes 404 78 81.5% (2)
P = .18 No 405 106 75% (2.2) P = .25 No 405 67 84% (1.9)

0 1 2 3 4 5 0 1 2 3 4 5

No. at risk: Time Since Random Assignment (years) No. at risk: Time Since Random Assignment (years)
Yes 404 340 319 287 258 204 Yes 404 356 335 313 290 232
No 405 366 338 302 282 219 No 405 376 356 332 310 247

C D
809 Patients 809 Patients
1.0 1.0
P = .94 No. of No. of P = .08 No. of No. of
0.9 Patients Relapses 5-year Cumulative Incidence 0.9 Patients Deaths 5-year Cumulative Incidence
Cumulative Incidence

Cumulative Incidence
0.8 Yes 404 72 17.7% (1.9) 0.8 Yes 404 38 9.5% (1.5)

0.7 No 405 72 17.2% (1.9) 0.7 No 405 25 5.7% (1.2)

0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1

0 1 2 3 4 5 0 1 2 3 4 5

No. at risk: Time Since Random Assignment (years) No. at risk: Time Since Random Assignment (years)
Yes 404 340 319 287 258 204 Yes 404 340 319 287 258 204
No 405 366 338 302 282 219 No 405 366 338 302 282 219

FIG 3. Overall analysis by random assignment with or without PEG-ASNASE (yes v no), ITT. (A) EFS; (B) OS; (C) cumulative incidence of relapse;
(D) cumulative incidence of deaths. EFS, event-free survival; OS, overall survival; PEG-ASNASE, pegylated L-asparaginase.

difference between the experimental versus control arm allo-HCT (9 of 74 (12.2%) versus 13 of 103 (12.6%) transplanted
(Appendix Table A1, model B). patients). Relapses were 64 (17.6%) and 80 (18.3%) in 363
patients treated versus 437 not treated with PEG-ASNASE.
When EFS was analyzed in BCP-ALL and T-ALL subgroups The 5-year EFS was 72.1% (2.4) in patients treated versus
(Figs 4A-4F), the incidence of death differed only in BCP-ALL 74.3% (2.1) in those not treated with PEG-ASNASE (P 5 .56),
cases: 10.6% (1.9) versus 5.8% (1.4) in the experimental versus with no significant difference in the CI of relapse (5-year
control arm (P 5 .04, Fig 4E). Estimates of the treatment figure: 17.5% [2.0] v 17.9% [1.9], P 5 .84) and of death (5-year
effect on EFS within subgroups defined by relevant prog- figure: 7.8% [1.4] v 6.0% [1.1], P 5 .46; Appendix Fig A3).
nostic features, as reported in the Appendix Figure A2, do not
show evidence of a differential effect in any subgroup. By immunophenotype, in both subgroups of BCP-ALL and
T-ALL, no significant differences were detected in EFS and
Outcome and Toxicity in the as-Treated Population cumulative incidence curves (Appendix Fig A4).

The secondary analysis on the as-treated population is The percentage of patients with medically important adverse
shown in the Appendix Table A3: deaths in phase IB in reactions during phase IB (life-threatening events included)
patients receiving or not receiving PEG-ASNASE were 6 was more than doubled in the PEG-ASNASE–treated (22.2%)
(1.6%) versus 2 (0.4%) and deaths in CR after phase IB versus not treated (8.9%) groups (Appendix Table A4). Of note,
were 22 (6.1%) versus 26 (6.0%), including deaths after an allergic reaction to PEG-ASNASE occurred in 15 patients

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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

A B
517 Patients 285 Patients
1.0 1.0

0.8 0.8
EFS (probability)

EFS (probability)
0.6 0.6

0.4 0.4
No. of No. of No. of No. of
Patients Events 5-year EFS Patients Events 5-year EFS
0.2 Yes 257 81 68.6% (3) 0.2 Yes 144 38 73.9% (3.7)
P = .12 No 260 68 75.3% (2.7) P = .86 No 141 37 74.6% (3.7)

0 1 2 3 4 5 0 1 2 3 4 5

No. at risk: Time Since Random Assignment (years) No. at risk: Time Since Random Assignment (years)
Yes 257 221 210 185 164 124 Yes 144 117 107 101 94 80
No 260 240 226 200 185 142 No 141 122 110 100 95 77

C D
517 Patients 285 Patients
1.0 1.0
P = .84 No. of No. of P = .99 No. of No. of
0.9 Patients Relapses 5-year Cumulative Incidence 0.9 Patients Relapses 5-year Cumulative Incidence
Cumulative Incidence

Cumulative Incidence
0.8 Yes 257 48 18.7% ( 2.5) 0.8 Yes 144 24 16.1% (3.1)
0.7 No 260 47 16.8% (2.4) No 141 24 17.6% (3.3)
0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1

0 1 2 3 4 5 0 1 2 3 4 5

No. at risk: Time Since Random Assignment (years) No. at risk: Time Since Random Assignment (years)
Yes 257 221 210 185 164 124 Yes 144 117 107 101 94 80
No 260 240 226 200 185 142 No 141 122 110 100 95 77

E F
517 Patients 285 Patients
1.0 1.0
P = .04 No. of No. of P = .92 No. of No. of
0.9 Patients Deaths 5-year Cumulative Incidence 0.9 Patients Deaths 5-year Cumulative Incidence
Cumulative Incidence

Cumulative Incidence

0.8 Yes 257 27 10.6% (1.9) 0.8 Yes 144 10 7% (2.1)

0.7 No 260 15 5.8% (1.4) 0.7 No 141 10 5.7% (2)

0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1

0 1 2 3 4 5 0 1 2 3 4 5

No. at risk: Time Since Random Assignment (years) No. at risk: Time Since Random Assignment (years)
Yes 257 221 210 185 164 124 Yes 144 117 107 101 94 80
No 260 240 226 200 185 142 No 141 122 110 100 95 77

FIG 4. Analysis by random assignment with or without PEG-ASNASE (yes v no), ITT in the two subgroups of BCP and T ALL. (A and B) EFS;
(C and D) cumulative incidence of relapse (rel); (E and F) cumulative incidence of deaths. BCP, B-cell precursor; EFS, event-free survival; ITT,
intention to treat; PEG-ASNASE, pegylated L-asparaginase; T ALL, T acute lymphoblastic leukemia.

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 Conter et al

(12 after dose 1, two after dose 2, and one after dose 3). Six mortality and slightly inferior EFS. However, seven fatal toxic
deaths occurred in phase IB in patients treated with at least one events in phase IB occurred very early after random assign-
dose of PEG-ASNASE, because of infection (n 5 4), pancreatitis ment, by chance all in the experimental arm, preventing
(n 5 1), and thrombosis (n 5 1), and 2 deaths occurred in these patients from starting the planned treatment with
patients not receiving PEG-ASNASE, one because of infection PEG-ASNASE. This finding explains the reduction in the
and the other because of hemophagocytic lymphohistiocytosis observed mortality difference in the as-treated analysis, as
(which started in phase IA and aggravated in phase IB). The shown by the cumulative incidence curves (Appendix Fig A3D
median duration of phase IB (and IQ range) in patients without [as-treated] and Fig 3D [ITT]).
fatal events was 62 (55-69) and 55 (49-62) days in patients
who did or did not receive PEG-ASNASE, respectively. In this trial, PEG-ASNASE was used weekly at high dose with
the aim of fully exploiting its potential benefit; of note, this
DISCUSSION treatment was associated with increased toxicity with more
than doubled medically important adverse reactions. Con-
The aim of this randomized trial was to assess if treatment sidering the pharmacokinetics and the pharmacodynamics
intensification during the Consolidation phase IB with of this drug, however, it may be argued that the full effect
PEG-ASNASE in HR patients would reduce MRD levels and could also be achieved with a less intensive schedule, which
increase EFS, with acceptable toxicity. might be associated with a decreased toxicity.

The PCR-MRD level at the end of consolidation was chosen Importantly, in both analyses, by ITT and by treatment
as the primary end point, considering its prognostic impact given, there is no evidence of benefit on reduction in relapses
on EFS in both BCP and T-ALL1,2 (although not yet validated by the additional therapy with PEG-ASNASE in phase IB, and
as a surrogate end point15). this finding is consistent with the very modest impact on
MRD levels (only in BCP-ALL) at the end of phase IB in the
Sample size calculation was reassessed during study experimental arm compared with the control arm. In addition,
conduction because of lower than expected percentage of this lack of efficacy may be due to the fact that the cumulative
patients with high MRD at TP2 in the control arm. This
exposure to PEG-ASNASE was quite high in patients also
expected level (33%) was calculated in the planning phase
randomly assigned to the control arm (2,500 IU/sqm 32 doses
on the AIEOP-BFM ALL 2000 study results. The lower rate
in induction and 36 doses in postconsolidation therapy).
observed in the study may be due to the substitution of
Overall, this experience is in line with that of the EORTC 58951
PEG-ASNASE for native E-coli L-asparaginase, the use of
randomized study, where patients at standard risk randomly
dexamethasone instead of prednisone in T-ALL PGR patients,
the addition of cyclophosphamide (day 8) in T-ALL PPR assigned for prolonged L-asparaginase exposure during
during the induction phase, and the adoption of day 15 BM phase IB had a higher risk of infection with similar outcomes
response as additional HR criteria. when compared with patients in the control arm.10

In this randomized study, results (ITT analysis) show in the L-asparagine depletion in the CSF in patients treated in this
experimental arm a very modest, not significant decrease in the protocol, despite the dosage of 2,500 IU/sqm, was found to
percentage of patients with high MRD, consistent with the be suboptimal.16 Not surprisingly thus, there was no dif-
nonsignificant difference in EFS between the two arms, making ference between arms in the rate of isolated CNS relapses.
it difficult to contribute to the validation of MRD as a surrogate
end point for final patient’s outcome. More in-depth, high Overall, outcomes in these HR patients can be considered
MRD (≥5 3 1024 at the end of consolidation) was detected in relatively satisfactory and comparable with those obtained with
13.9% versus 17.0% of patients in the experimental versus other intensive therapeutic strategies.17-20 However, this early
control arm. Subanalyses by immunophenotype showed that intensification of chemotherapy with the four additional doses
the decrease occurred only in BCP-ALL and was more evident in of PEG-ASNASE in HR patients has not resulted in improved
those patients with high MRD levels at TP1. In general, there is disease control or clinical outcome. This suggests that within
no proof for efficacy in disease control by additional therapy intensive treatment protocols, further improvements are dif-
with PEG-ASNASE, as assessed by MRD response. The per- ficult to achieve with intensification of antileukemic therapy, as
centage of patients eligible to allo-HCT was similar in both other recent studies have shown.22
arms, which is approximately 26%.
This points to the need of exploring innovative approaches in
The interpretation of results in terms of EFS is complicated HR patients, as it is being pursued in current studies, in-
by toxicity. The analysis by ITT on EFS suggests that the cluding the AIEOP-BFM ALL 2017 study, where for BCP-ALL,
additional administration of intensive PEG-ASNASE in phase the potential benefit of proteasome inhibitors and of specific
IB was associated, although not significantly, with increased monoclonal antibodies is being investigated.

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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

AFFILIATIONS EQUAL CONTRIBUTION

1
Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, V.C. and M.G.V share the first author position. A.B. and M.S. share the
Italy last author position.
2
School of Medicine and Surgery, University of Milano-Bicocca, Milan,
Italy CLINICAL TRIAL INFORMATION
3
Biostatistics and Clinical Epidemiology, Fondazione IRCCS San
NCT01117441
Gerardo dei Tintori, Monza, Italy
4
Department of Pediatrics I, Pediatric Hematology/Oncology, ALL-BFM
Study Group, Christian Albrechts University Kiel and University Hospital
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS
Schleswig-Holstein, Campus Kiel, Kiel, Germany
OF INTEREST
5
Department of Pediatric Hematology/Oncology, Hannover Medical Disclosures provided by the authors are available with this article at DOI
School, Hannover, Germany https://doi.org/10.1200/JCO.23.01388.
6
Department of Pediatric Hematology and Oncology, St Anna Children’s
Hospital, Medical University of Vienna, Vienna, Austria DATA SHARING STATEMENT
7
St Anna Children’s Cancer Research Institute, Vienna, Austria
8 Deidentified data and the trial protocol are available to qualified
Department of Pediatric Haematology and Oncology, 2nd Faculty of
researchers who submit an in-scope proposal. The data will be available
Medicine, Charles University in Prague and Motol University Hospital,
from the authors of the publication to ensure that data are used in
Prague, Czech Republic
9 accordance with the informed consent. Data are provided for the sole
University Children Hospital Zurich, Department of Oncology, Zurich,
use of the approved request (tertiary dissemination will not be
Switzerland
10 permitted). To gain access, data requesters will need to sign a data
The Cancer Centre for Children, The Children’s Hospital at Westmead,
sharing agreement.
Sydney, NSW, Australia
11
Department of Pediatric Hematology-Oncology, Schneider Children’s
Medical Center, Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel
AUTHOR CONTRIBUTIONS
12
Department of Pediatric Hematology and Oncology, IRCCS Ospedale Conception and design: Valentino Conter, Maria Grazia Valsecchi,
Bambino Gesù, Rome, Catholic University of the Sacred Heart, Rome, Gunnar Cario, Martin Zimmermann, Andishe Attarbaschi, Jan Stary,
Italy Felix Niggli, Luciano Dalla Pozza, Franco Locatelli, Anja Möricke,
13
Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Shai Izraeli, Andrea Biondi, Martin Schrappe
Australia Provision of study materials or patients: Gunnar Cario, Andishe
14
Pediatrics, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy Attarbaschi, Jan Stary, Felix Niggli, Luciano Dalla Pozza, Sarah Elitzur,
15
Department of Paediatric Hematology and Oncology, University Franco Locatelli, Anja Möricke, Gernot Engstler, Petr Smisek,
Hospital Muenster, Muenster, Germany Nicole Bodmer, Draga Barbaric, Shai Izraeli, Carmelo Rizzari, Joachim
16
Pediatric Hematology, Oncology, and Stem Cell Transplant Division, Boos, Barbara Buldini, Massimo Zucchetti, Arend von Stackelberg,
Maternal and Child Health Department, Padua University, Padua, Italy Cristina Matteo, Thomas Lehrnbecher, Claudia Lanvers-Kaminsky,
17
Department of Oncology, Laboratory of Cancer Pharmacology, Giovanni Cazzaniga, Bernd Gruhn, Andrea Biondi, Martin Schrappe
IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy Collection and assembly of data: Maria Grazia Valsecchi, Martin
18
Department of Pediatric Oncology and Hematology, Zimmermann, Daniela Silvestri, Anja Möricke
Charité - Universitätsmedizin Berlin, Berlin Institute of Health at Data analysis and interpretation: All authors
Charité - Universitätsmedizin Berlin, Berlin, Germary Manuscript writing: All authors
19
Department of Pediatrics, Division of Hematology and Oncology, Final approval of manuscript: All authors
Goethe University Frankfurt, Frankfurt am Main, Germany Accountable for all aspects of the work: All authors
20
Department of Pediatrics, Jena University Hospital, Jena, Germany
ACKNOWLEDGMENT
CORRESPONDING AUTHOR The authors thank all participating institutions, physicians, and patients
Valentino Conter, MD, Tettamanti Center, Fondazione IRCCS San for their support to the study and dedicate this work to the memory of
Gerardo dei Tintori, via Pergolesi, 33, 20900 Monza, Italy; Prof. Giuseppe Basso. The authors would like to acknowledge the
e-mail: [email protected]. members of the Data and Safety Monitoring Committee for their advice
during study conduction (J. Nachman†, A. Ganser, R. Foà, J. Boyett).

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newly diagnosed acute lymphoblastic leukemia: Results from a randomized study—Dana-Farber Cancer Institute ALL Consortium Protocol 00-01. J Clin Oncol 31:1202-1210, 2013

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8. Nachman J, Sather HN, Gaynon PS, et al: Augmented Berlin-Frankfurt-Munster therapy abrogates the adverse prognostic significance of slow early response to induction chemotherapy for
children and adolescents with acute lymphoblastic leukemia and unfavorable presenting features: A report from the Children’s Cancer Group. J Clin Oncol 15:2222-2230, 1997
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10. Mondelaers V, Suciu S, De Moerloose B, et al: Prolonged versus standard native E. coli asparaginase therapy in childhood acute lymphoblastic leukemia and non-Hodgkin lymphoma: Final results
of the EORTC-CLG randomized phase III trial 58951. Haematologica 102:1727-1738, 2017
11. Dworzak MN, Buldini B, Gaipa G, et al: AIEOP-BFM consensus guidelines 2016 for flow cytometric immunophenotyping of Pediatric acute lymphoblastic leukemia. Cytometry B Clin Cytom 94:
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12. Basso G, Veltroni M, Valsecchi MG, et al: Risk of relapse of childhood acute lymphoblastic leukemia is predicted by flow cytometric measurement of residual disease on day 15 bone marrow. J Clin
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13. Flohr T, Schrauder A, Cazzaniga G, et al: Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements
in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia. Leukemia 22:771-782, 2008
14. Rizzari C, Möricke A, Valsecchi MG, et al: Incidence and characteristics of hypersensitivity reactions to PEG-asparaginase observed in 6136 children with acute lymphoblastic leukemia enrolled in
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pky069, 2018
16. Rizzari C, Lanvers-Kaminsky C, Valsecchi MG, et al: Asparagine levels in the cerebrospinal fluid of children with acute lymphoblastic leukemia treated with pegylated-asparaginase in the induction
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17. Vora A, Goulden N, Mitchell C, et al: Augmented post-remission therapy for a minimal residual disease-defined high-risk subgroup of children and young people with clinical standard-risk and
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18. Toft N, Birgens H, Abrahamsson J, et al: Results of NOPHO ALL2008 treatment for patients aged 1-45 years with acute lymphoblastic leukemia. Leukemia 32:606-615, 2018
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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Four Additional Doses of PEG-L-Asparaginase During the Consolidation Phase in the AIEOP-BFM ALL 2009 Protocol Do Not Improve Outcome and
Increase Toxicity in HR ALL: Results of a Randomized Study

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless
otherwise noted. Relationships are self-held unless noted. I 5 Immediate Family Member, Inst 5 My Institution. Relationships may not relate to the
subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or
ascopubs.org/jco/authors/author-center.

Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open
Payments).

Valentino Conter Joachim Boos

Consulting or Advisory Role: Amgen Research Funding: Servier (Inst)
Travel, Accommodations, Expenses: Jazz Pharmaceuticals
Barbara Buldini
Gunnar Cario Speakers’ Bureau: Beckman Coulter, Becton Dickinson, Amgen
Honoraria: Amgen (Inst) Travel, Accommodations, Expenses: Beckman Coulter, Becton
Consulting or Advisory Role: Jazz Pharmaceuticals (Inst) Dickinson, Amgen
Travel, Accommodations, Expenses: Jazz Pharmaceuticals (Inst)
Arend von Stackelberg
Andishe Attarbaschi Consulting or Advisory Role: Amgen, Novartis, Jazz Pharmaceuticals,
Honoraria: Jazz Pharmaceuticals, Amgen, Novartis Miltenyi Biotec
Consulting or Advisory Role: Jazz Pharmaceuticals, Amgen, Novartis,
Takeda Science Foundation Thomas Lehrnbecher
Travel, Accommodations, Expenses: Jazz Pharmaceuticals Consulting or Advisory Role: Gilead Sciences, Merck Sharp & Dohme,
Roche, Mundipharma, Pfizer, EUSA Pharma, AstraZeneca
Sarah Elitzur Speakers’ Bureau: Gilead Sciences, Merck Sharp & Dohme, Sanofi, EUSA
Honoraria: Medison Pharma, Pfizer
Consulting or Advisory Role: Amgen, Jazz Pharmaceuticals Research Funding: Gilead Sciences
Travel, Accommodations, Expenses: Gilead Sciences, Merck Sharp &
Franco Locatelli Dohme
Honoraria: Miltenyi Biotec, Bluebird Bio, SOBI, Amgen, Novartis
Consulting or Advisory Role: Amgen, Novartis, Pfizer Claudia Lanvers-Kaminsky
Honoraria: Clinigen Group (Inst)
Anja Möricke Travel, Accommodations, Expenses: Servier
Consulting or Advisory Role: Clinigen Group, BTG
Andrea Biondi
Shai Izraeli Consulting or Advisory Role: CoImmune, Galapagos NV, BMS
Honoraria: Foundation Medicine Speakers’ Bureau: Amgen, Novartis
Speakers’ Bureau: Oncotest/Rhenium Research Funding: CoImmune

Carmelo Rizzari Martin Schrappe

Honoraria: Servier, Jazz Pharmaceuticals, Clinigen Group, Amgen, SERB Consulting or Advisory Role: Servier, Jazz Pharmaceuticals
Consulting or Advisory Role: Servier, Jazz Pharmaceuticals, Clinigen Speakers’ Bureau: Jazz Pharmaceuticals
Group, SERB Research Funding: Servier (Inst)
No other potential conflicts of interest were reported.

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 Conter et al

APPENDIX

TABLE A1. Results of the Logistic Model on the Primary End Point (MRD day 178) and of the Cox Model on Secondary End Point (EFS), ITT Analysis

Model A Model B
Logistic Model Cox Model

Covariates OR P 95% CI Hazard Ratio P 95% CI

Randomized treatment
Without PEG-ASNASE 1 1
With PEG-ASNASE 0.77 .22 0.51 to 1.17 1.05 .73 0.78 to 1.42
Sex
Male 1 1
Female 0.94 .77 0.60 to 1.46 1.07 .69 0.78 to 1.45
Age at diagnosis, years
1-9 1 1
10-17 1.68 .02 1.10 to 2.55 1.38 .04 1.02 to 1.86
Immunophenotype
BCP-ALL 1 1
T ALL 1.17 .56 0.69 to 1.97 0.62 .02 0.42 to 0.93
WBC, 3109/L, No. (%)
<100 1 1
≥100 1.94 .007 1.20 to 3.15 1.73 .002 1.21 to 2.45
PDN response
Good 1 1
Poor 0.83 .44 0.51 to 1.34 1.13 .50 0.79 to 1.51
FCM-MRD day 115
<10% — — — 1
≥10% 1.22 .32 0.83 to 1.80
PCR-MRD day 133
Negative or positive <5 3 1024 — — — 1
Positive ≥5 3 1024 1.41 .06 0.99 to 2.00

Abbreviations: BCP-ALL, B-cell precursor-ALL; EFS, event-free survival; FCM-MRD, flow cytometry-minimal residual disease; ITT, intention to treat;
MRD, minimal residual disease; OR, odds ratio; PCR-MRD, polymerase chain reaction minimal residual disease; PDN, prednisone; PEG-ASNASE,
pegylated L-asparaginase.

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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

TABLE A2. Primary End Point Based on PCR-MRD at TP2 in Randomly Assigned Patients With PCR-MRD at TP1 ≥5 3 1024, With the Available Data
by ITT and by the Immunophenotype Subgroup

Total BCP-ALL T ALL

With PEG- Without PEG- With PEG- Without PEG- With PEG- Without PEG-
PCR-MRD at Day 178 ASNASE ASNASE ASNASE ASNASE ASNASE ASNASE
No. of Patients 236 217 130 127 105 87
Negative or positive NQ, No. 139 (58.9) 120 (55.3) 76 (58.4) 73 (57.5) 62 (59.0) 45 (51.7)
(%)
Positive <5 3 1024, No. (%) 47 (19.9) 37 (17.0) 27 (20.8) 16 (12.6) 20 (19.1) 21 (24.1)
Positive ≥5 3 1024, No. (%) 50 (21.2) 60 (27.7) 27 (20.8) 38 (29.9) 23 (21.9) 21 (24.1)

Abbreviations: BCP-ALL, B-cell precursor-ALL; ITT, intention to treat; NQ, not quantifiable; PCR-MRD, polymerase chain reaction minimal residual
disease; PEG-ASNASE, pegylated L-asparaginase.

TABLE A3. Events After Random Assignment (secondary end point): “As-Treated” Analysis

Status With PEG-ASNASE Without PEG-ASNASE Total

Randomly assigned patients, No. 363 437 800
Resistant by protocol, No. (%) 2 (0.6) 1 (0.2) 3 (0.4)
Deaths, No. (%) 28 (7.7) 28 (6.4) 56 (7.0)
Phase IB 6 (1.6) 2 (0.4) 8 (1.0)
After phase IB (CHEMO) 13 (3.6) 13 (3.0) 26 (3.2)
After allo-HCT 9 (2.5) 13 (3.0) 22 (2.7)
Relapses, No. (%) 64 (17.6) 80 (18.3) 144 (18.0)
BM isolated 34 (9.3) 54 (12.3) 88 (11.0)
BM combined 14 (3.9) 12 (2.7) 26 (3.2)
CNS isolated 11 (3.0) 9 (2.1) 20 (2.5)
TESTIS isolated 1 (0.3) 2 (0.5) 3 (0.4)
Other 4 (1.1) 3 (0.7) 7 (0.9)
Second malignant neoplasm, No. (%) 8 (2.2) 8 (1.8) 16 (2.0)
Alive in CCR, No. (%) 261 (71.9) 320 (73.3) 581 (72.6)

HCT Status With PEG-ASNASE Without PEG-ASNASE Total

Allo-HCT eligibility, No. (%) 96 (26.4) 116 (26.5) 212 (26.5)
Allo-HCT performed, No. (%) 74 (20.4) 103 (23.6) 177 (22.1)

NOTE. It excludes events before day 140, that is, time of start of randomized treatment.
Abbreviations: allo-HCT, allogeneic-hematopoietic cell transplantation; BM, bone marrow; CCR, continuous complete remission; PEG-ASNASE,
pegylated L-asparaginase.

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 Conter et al

TABLE A4. Medically Important AR During Consolidation IB in the “As-Treated” Population Including 363 Patients in Arm With PEG-ASNASE,
Receiving at Least One Dose and to 437 Patients in Arm Without PEG-ASNASE

Life-Threateninga AR

AR (including life-threateninga AR) Nonfatal Fatal

With PEG- Without PEG- With PEG- Without PEG- With PEG- Without PEG-
Adverse Reactions ASNASE ASNASE ASNASE ASNASE ASNASE ASNASE
No. of patients with AR 81 (22.2) 39 (8.9) 14 (3.9) 5 (1.1) 6 (1.6) 2 (0.4)
P <.001 .002b
No. of AR 93 (25.5) 41 (9.3) 15 (4.1) 5 (1.1) 6 (1.6) 2 (0.4)
Infectious 32 (8.8) 17 (3.9) 8 (2.2) 3 (0.7) 4 (1.1) 1 (0.2)
Pancreatic 18 (4.9) 2 (0.5) 2 (0.6) 0 1 (0.3) 0
Thrombotic (including 10 (2.7) 4 (0.9) 2 (0.6) 0 1 (0.3) 0
cerebral)
Hepatic 7 (1.9) 4 (0.9) 1 (0.3) 0 0 0
Neurologic/psychologic 10 (2.7) 10 (2.3) 2 (0.6) 1 (0.2) 0 0
Gastrointestinal 1 (0.3) 0 0 0 0 0
Metabolic/endocrine 0 1 (0.2) 0 1 (0.2) 0 0
Drug-related allergic 15 (4.1) 0 0 0 0 0
reactions
Skeletal 0 1 (0.2) 0 0 0 0
Other/not classifiable 0 2 (0.4) 0 0 0 1 (0.2)c

NOTE. The AR here reported were predefined in the study protocol “Medically important AR of interest.”
Abbreviations: AR, adverse reactions; PEG-ASNASE, pegylated L-asparaginase.
a
An AR was considered life-threatening if its occurrence placed the patient at immediate risk of death.
b
P value for the comparison of proportion of patients with life-threatening events (nonfatal and fatal together).
c
This patient died in phase IB (after day 40) for HLH which started in phase IA and aggravated in phase IB.

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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

A
General Outline of High Risk in AIEOP-BFM ALL 2009 Study

IACPM
IB HR HR HR
HR RHR
1 2 3 III III III CP
IB
IA FLA-DNX

Allogeneic HSCT 104

Week 1 6 10 12 15 18 2122 24 27 30 31 38 41
PEG-ASNASE 2,500 IU/SQM/DOSE

B
Consolidation Phase IB in AIEOP-BFM ALL 2009 Study

PEG L-ASP p.i. (2 hr) 2,500 IU/m2/dose Only given in the

ONCASPAR4 (max. 3,750 IU) experimental arm

CPM p.i. (1 hr) 1,000 mg/m2/dose

ARA-C i.v. 75 mg/m2/dose

6-MP p.o. (28 d) 60 mg/m2/day

MTXIT
Age-adjusted dose:
1 to <2 years: 8 mg
2 to <3 years: 10 mg
t3 years: 12 mg Days 36 43 50 57 64

FIG A1. General outline of the HR treatment showing random assignment (Fig 1A) and treatment details
of phase IB (Fig 1B). HR, high risk; HSCT, hematopoietic stem-cell transplantation; PEG-ASNASE,
pegylated L-asparaginase.

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 Conter et al

5-Year EFS (SE) Experimental

With Without Hazard Ratio No. of Patients
PEG-ASNASE PEG-ASNASE (95% CI) (No. of events) Better Worse

Male 70.8 (2.9) 76.0 (2.7) 1.19 (0.85-1.65) 517 (139)

Female 69.7 (3.9) 73.3 (3.7) 1.21 (0.80-1.85) 292 (87)
1-9 years 72.6 (2.9) 77.7 (2.7) 1.22 (0.86-1.73) 500 (128)
10-17 years 66.8 (3.9) 70.3 (3.9) 1.16 (0.78-1.72) 309 (98)
WBC <100 74.6 (2.7) 77.2 (2.5) 1.08 (0.78-1.50) 564 (141)
WBC ≥100 61.4 (4.4) 69.6 (4.3) 1.42 (0.92-2.18) 245 (85)
PGR 72.2 (3.3) 75.1 (3.2) 1.09 (0.74-1.59) 384 (106)
PPR 69.0 (3.3) 74.7 (3.1) 1.28 (0.89-1.84) 415 (117)
FCM15 <10% 73.9 (4.2) 77.7 (4.3) 1.29 (0.73-2.25) 211 (50)
FCM15 ≥10% 70.2 (2.8) 74.0 (2.6) 1.12 (0.83-1.52) 573 (167)
PCR33 low 77.1 (3.8) 79.7 (3.3) 1.10 (0.67-1.82) 285 (61)
PCR33 high 70.1 (3.0) 71.7 (3.1) 1.02 (0.73-1.43) 457 (139)
BCP 68.6 (3.0) 75.3 (2.7) 1.29 (0.93-1.78) 517 (149)
T ALL 73.9 (3.7) 74.6 (3.7) 1.04 (0.66-1.64) 285 (75)
Overall 70.4 (2.3) 75.0 (2.2) 1.20 (0.92-1.55) 809 (226)

0.00 1.00 2.00 3.00

FIG A2. Analysis of treatment effect (ITT) within subgroups and on the overall sample with the estimated hazard
ratio from a Cox model (univariate analysis). BCP, B-cell precursor; EFS, event-free survival; FCM, flow cytometry;
ITT, intention to treat; L-Asp, L-asparaginase; PCR, polimerase chain reaction; PGR, prednisone good responder;
PPR, prednisone poor response.

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 Additional PEG-ASNASE During Consolidation in Childhood HR-ALL

A 800 Patients
B 800 Patients
1.0 1.0

0.8 0.8
EFS (probability)

OS (probability)
0.6 0.6

0.4 0.4
No. of No. of No. of No. of
Patients Events 5-year EFS Patients Deaths 5-Year OS
0.2 0.2
Yes 363 102 72.1% (2.4) Yes 363 62 83.9% (2)
P = .56 No 437 117 74.3% (2.1) P = .99 No 437 76 83% (1.8)

0 1 2 3 4 5 0 1 2 3 4 5

Time Since Random Assignment (years) Time Since Random Assignment (years)
No. at risk: No. at risk:
Yes 363 311 292 263 239 190 Yes 363 325 308 287 269 217
No 437 393 363 324 299 232 No 437 405 381 356 329 261

C D
800 Patients 800 Patients
1.0 P = .84 No. of No. of 5-Year Cumulative
1.0 P = .46 No. o No. of 5-Year Cumulative
0.9 Patients Relapses Incidence 0.9 Patients Deaths Incidence
Cumulative Incidence

Cumulative Incidence
Yes 363 64 17.5% (2) Yes 363 28 7.8% (1.4)
0.8 0.8
No 437 80 17.9% (1.9) No 437 28 6% (1.1)
0.7 0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1

0 1 2 3 4 5 0 1 2 3 4 5

Time Since Random Assignment (years) Time Since Random Assignment (years)
No. at risk: No. at risk:
Yes 363 311 292 263 239 190 Yes 363 311 292 263 239 190
No 437 393 363 324 299 232 No 437 393 363 324 299 232

FIG A3. Overall analysis by random assignment to additional PEG-ASNASE or not (yes v no), in the as treated population. (A) EFS, (B) OS, (C)
cumulative incidence of relapse, and (D) cumulative incidence of deaths. EFS, event-free survival; OS, overall survival; PEG-ASNASE, pegylated L-
asparaginase.

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 Conter et al

A 512 Patients
D 281 Patients
1.0 1.0
EFS (probability)

EFS (probability)
0.8 0.8

0.6 0.6

0.4 0.4
No. of No. of No. of No. of
Patients Events 5-Year EFS Patients Events 5-Year EFS
0.2 0.2
Yes 233 68 71.1% (3) Yes 127 33 74.3% (3.9)
P = .92 No 279 77 73.7% (2.7) P = .81 No 154 39 75.4% (3.5)

0 1 2 3 4 5 0 1 2 3 4 5

Time Since Random Assignment (years) Time Since Random Assignment (years)
No. at risk: No. at risk:
Yes 233 205 195 173 156 119 Yes 127 104 95 89 83 71
No 279 255 240 211 192 147 No 154 134 121 111 105 85

B E
512 Patients 281 Patients
1.0 P = .57 No. of No. of 5-Year Cumulative
1.0 P = .59 No. of No. of 5-Year Cumulative
0.9 Patients Relapses Incidence 0.9 Patients Relapses Incidence
Cumulative Incidence

Cumulative Incidence
0.8 Yes 233 41 17.6% (2.6) 0.8 Yes 127 23 17.5% (3.4)
No 279 54 18.3% (2.4) No 154 25 16.8% (3.1)
0.7 0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1

0 1 2 3 4 5 0 1 2 3 4 5

Time Since Random Assignment (years) Time Since Random Assignment (years)
No. at risk: No. at risk:
Yes 233 205 195 173 156 119 Yes 127 104 95 89 83 71
No 279 255 240 211 192 147 No 154 134 121 111 105 85

C F
512 Patients 281 Patients
1.0 P = .20 No. of No. of 5-Year Cumulative
1.0 P = .38 No. of No. of 5-Year Cumulative
0.9 Patients Deaths Incidence 0.9 Patients Deaths Incidence
Cumulative Incidence

Cumulative Incidence

0.8 Yes 233 21 9.1% (1.9) 0.8 Yes 127 6 4.8% (1.9)
No 279 17 6.1% (1.4) No 154 11 5.9% (1.9)
0.7 0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1

0 1 2 3 4 5 0 1 2 3 4 5

Time Since Random Assignment (years) Time Since Random Assignment (years)
No. at risk: No. at risk:
Yes 233 205 195 173 156 119 Yes 127 104 95 89 83 71
No 279 255 240 211 192 147 No 154 134 121 111 105 85

FIG A4. Analysis by random assignment to additional PEG-ASNASE or not (yes v no), in the as treated population in the subgroups of BCP
and T ALL. (A and D) EFS, (B and E) cumulative incidence of relapse, and (C and F) cumulative incidence of deaths. EFS, event-free
survival; PEG-ASNASE, pegylated L-asparaginase.

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