Clofarabine Increases the Eradication of Minimal Residual Disease Of

Home , Clofarabine

Clofarabine increases the eradication of minimal residual disease of primary B-precursor acute lymphoblastic leukemia compared to high-dose cytarabine without improvement of outcome Results from the randomized clinical trial 08-09 of the Cooperative Acute Lymphoblastic Leukemia Study Group by Gabriele Escherich, Udo zur Stadt, Arndt Borkhardt, Dagmar Dilloo, Jörg Faber, Tobias Feuchtinger, Thomas Imschweiler, Norbert Jorch, Arnulf Pekrun, Irene Schmid, Franziska Schramm, Michale Spohn, Martin Zimmermann, and Martin A. Horstmann

Received: June 1, 2021. Accepted: July 16, 2021.

Citation: Gabriele Escherich, Udo zur Stadt, Arndt Borkhardt, Dagmar Dilloo, Jörg Faber,Tobias Feuchtinger, Thomas Imschweiler, Norbert Jorch, Arnulf Pekrun, Irene Schmid, Franziska Schramm, Michale Spohn, Martin Zimmermann, and Martin A. Horstmann. Clofarabine increases the eradication of minimal residual disease of primary B-precursor acute lymphoblastic leukemia compared to high-dose cytarabine without improvement of outcome Results from the randomized clinical trial 08-09 of the Cooperative Acute Lymphoblastic Leukemia Study Group. Haematologica. 2021 Aug 5. doi:10.3324/haematol.2021.279357. [Epub ahead of print]

Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors final approval the final version of the manuscript will then appear in a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. Clofarabine increases the eradication of minimal residual disease of primary B- precursor acute lymphoblastic leukemia compared to high-dose cytarabine without improvement of outcome Results from the randomized clinical trial 08-09 of the Cooperative Acute Lymphoblastic Leukemia Study Group

Gabriele Escherich1, Udo zur Stadt1, Arndt Borkhardt2, Dagmar Dilloo3, Jörg Faber4,

Tobias Feuchtinger5, Thomas Imschweiler6, Norbert Jorch7, Arnulf Pekrun8, Irene

Schmid5, Franziska Schramm1, Michael Spohn 1,9,10, Martin Zimmermann11, Martin A

Horstmann1,9

1Clinic of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

2Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty Duesseldorf, Duesseldorf, Germany;

3Department of Pediatric Hematology/Oncology, University Hospital Bonn, Bonn, Germany.

4Department of Pediatric Hematology/Oncology, University Hospital Mainz, Mainz, Germany.

5Dr. von Hauner Children's Hospital, Ludwig Maximilian University, Munich, Germany.

6Department of Pediatric Hematology and Oncology, Helios Hospital, Krefeld, Germany.

7Department of Pediatric Hematology and Oncology, Protestant Hospital of Bethel Foundation, Bielefeld, Germany.

8Department of Pediatric Hematology and Oncology, Hospital Bremen-Mitte, Germany.

9 Research Institute Children’s Cancer Center Hamburg, Germany

10 Bioinformatics Core Unit, University Medical Center Hamburg, Germany

11 Department of Pediatric Hematology and Oncology, Medical School Hannover, Hannover, Germany

Abstract word count: 250 words

Manuscript word count: 2492 words

Figures: 4

Tables: 2

References: 24

Supplementary files: 1

Corresponding authors: Gabriele Escherich or Martin A. Horstmann, University Medical Center Eppendorf, Clinic of Pediatric Hematology and Oncology, Martinistrasse 52, 20246 Hamburg, Germany Phone: +49 40 74105 2580 Fax: +49 40 74105 8101 Mail: [email protected] or [email protected]

Running title: lymphoblastic leukaemia, paediatric, clofarabine

ClinicalTrials.gov identifier:: GPOH-CoALL 08-09 EU-21076 /NCT 0122 8331 https://clinicaltrials.gov/ct2/show/NCT01228331

Acknowledgements

Genzyme/Sanofi provided the investigational drug clofarabine. We thank Kseniya

Bakharevich for her assistance in collecting and interpreting the data. We gratefully acknowledge all patients, their families and care providers who participated in this study.

Finally, we thank all the clinicians, as well as diagnostics and research personnel who were actively involved in this clinical trial.

Disclosues

All authors declare no competing interests.

Contributions

MZ, MAH and GE designed the study with input from FS and UzS. DD, JF, TF, TI, NJ,

AP, IS and FS recruited patients. MAH, GE and FS collected, analysed, and interpreted data.

MZ did the statistical analysis. MS analysed data. All authors provided input for the data interpretation, reviewed, and critically revised the content of the manuscript. All authors approved the final version for submission.

Abstract

Novel treatment strategies are needed to improve cure for all children with acute lymphoblastic leukemia. To this end, we investigated the therapeutic potential of clofarabine in primary acute lymphoblastic leukemia in trial CoALL 08-09. The primary study objective was the minimal residual disease (MRD)-based comparative assessment of cytotoxic efficacies of clofarabine 5x40 mg/m2 versus high-dose cytarabine (HIDAC)

4x3g/m2, both in combination with PEG-ASP 2500 IU/m2 as randomized intervention in early consolidation. The secondary objective was an outcome analysis focused on treatment-arm dependence and MRD after randomized intervention. In B-cell precursor

(BCP)-ALL, eradication of MRD was more profound after clofarabine compared to cytarabine, with 93 vs 79 of 143 randomized patients per arm reaching MRD-negativity

(Chi-square test P=.03, left-sided P(Fisher’s exact test)=.04). MRD status of BCP-ALL after randomized intervention maintained its prognostic relevance, with a significant impact on event-free survival (EFS) and relapse rate. However, no difference in outcome regarding EFS and overall survival (OS) between randomized courses was observed (5- year EFS: clofarabine 85.7, SE=4.1 vs HIDAC 84.8, SE=4.7 (P=.96); OS: 95.7, SE=1.9 vs 92.2, SE=3.2 (P=.59)), independent of covariates or overall risk strata. Severe toxicities between randomized and subsequent treatment elements were also without significant difference.

In conclusion, clofarabine/PEG-ASP is effective and safe, but greater cytotoxic efficacy of clofarabine compared to HIDAC did not translate into improved outcomes indicating a lack of surrogacy of post-intervention MRD at the trial level as opposed to the patient

level, which hampers a broader implementation of this regimen in the frontline treatment of ALL.

Word count: 250

Introduction

The prevention of relapse without increasing toxicity is a challenging goal of frontline treatment in acute lymphoblastic leukemia (ALL), which is unlikely to be achieved by recombination or intensification of established chemotherapeutic agents. Beside immunotherapeutical approaches, novel compounds must be probed to prevent the development of resistant clones or to efficiently overcome those that already exist.

To this end, we evaluated clofarabine as one of the latest chemotherapeutic drugs to receive authoritative approval for the treatment of relapsed/refractory ALL in childhood.

Clofarabine is a second-generation purine nucleoside analogue that combines the positive characteristics of ﬁrst-generation purine nucleosides ﬂudarabine and cladribine by retaining 2-halogenated adenines, resulting in improved resistance against deamination and phosphorolysis.1-3 Several studies have been launched which scrutinized clofarabine in combination with other cytostatic drugs as second- or third-line therapy, or as a bridging regimen to hematopoietic stem cell transplantation.4-6

In Children’s Oncology Group (COG) trial AALL1131, clofarabine was administered in combination with etoposide and cyclophosphamide, which were associated with severe infections and persistent myelotoxicity leading to premature closure of the experimental clofarabine arm.7

To assess the value of the frontline usage of clofarabine, the cooperative acute lymphoblastic leukemia study group (CoALL) conducted a sequential phase II/III trial embedded into the CoALL 08-09 regimen for newly diagnosed ALL patients for whom end-of-induction (EOI) MRD imposed a greater risk of relapse.

During the non-randomized phase II, all eligible patients with quantifiable EOI MRD received the combination of clofarabine 5 x 40 mg/m2 and pegylated asparaginase

(PEG-ASP) 2500 IU/m2 as early consolidation treatment. The results were compared to a high-dose cytarabine (HIDAC)/PEG-ASP control group in predecessor trial CoALL 03-

07. Combined administration of clofarabine and PEG-ASP was feasible and exhibited acceptable toxicities without unexpected severe side effects.8

Herein, we describe the results of the subsequent phase III trial within CoALL 08-09, comparing the efficacy and tolerability of clofarabine/PEG-ASP versus HIDAC/PEG-ASP at early consolidation in a randomized fashion.

Methods

Study design and patients

CoALL 08-09 was a multi-center, randomized trial for patients under the age of 18 years with a confirmed diagnosis of acute B- or T-cell precursor leukemia. Accrual was open from 1 October 2010 to 31 December 2019. The study was approved by competent ethics boards (Table S1) and conducted in accordance with the Helsinki Declaration of

2008. The efficacy of clofarabine/PEG-ASP was compared with HIDAC/PEG-ASP in a randomized fashion as a primary study objective. An additional randomization of anthracyclines in delayed intensification was conducted from 2010 to 2016 with the primary objective of comparing toxicities.9

Stratification and treatment

All patients received the same three-drug induction with 4 weekly doses of daunorubicin

(36 mg/m2) and vincristine (1.5 mg/m2) along with oral methylprednisolone (60 mg/m2) over 28 days and a single dose of age-adapted intrathecal methotrexate. BCP-ALL with a discernible, but non-quantifiable, or quantifiable EOI MRD and T-ALL with ≥10-3 EOI

MRD were eligible for randomization, receiving either clofarabine 5 x 40 mg/m2 or

HIDAC 4 x 3 g/m2 in combination with PEG-ASP 2500 IU/m2 as the first or second course of consolidation in the treatment of BCP-ALL or T-ALL, respectively (Figure 1A).

Further treatment was administered according to respective strata (Figure 1B). By protocol, enrolled patients who achieved MRD-negativity at the end of induction or inversely showed an induction failure were not eligible for randomization (supplemental information).

Randomization

The randomization was performed by the coordinating trial center after stratification had been finalized according to EOI MRD status. Each stratum (HR patients were sub- divided according to immunophenotype) underwent independent randomization on the basis of randomly permuted blocks to avoid imbalances within risk strata.

Analysis of minimal residual disease

Real-time quantitative PCR analyses were performed targeting immunoglobulin heavy chain (IGH) and T-cell receptor (TCR) gene rearrangements to assess MRD. Data were

interpreted according to the guidelines developed by the European Study Group for

MRD detection in ALL (EuroMRD ALL).10

Statistics

The probability of event-free (pEFS) and overall survival (pOS) was estimated using the

Kaplan-Meier method and compared between subgroups using the log-rank test.11

Cumulative incidence functions of isolated CNS or any (isolated and combined) CNS relapse, as well as testicular relapse, treatment-related secondary malignancies and toxicity-related death were calculated using the Kalbfleisch and Prentice method and compared using Gray’s test.12 A Chi-square test, a Fisher’s exact test, and Spearman’s rank correlation analyses were applied to compare the distribution of parameters between subgroups and correlation between parameters.13 A Chi-square test was applied to determine the difference in the rate of MRD-positive patients, as provided in the study protocol. This was complemented by a one-sided Fisher’s exact test and a

Cochran-Armitage trend test, the latter of which compared the trend in MRD values between randomized groups.14

The status of patients was monitored annually. The database was newly updated (1

December 2020) before being used for analysis. Analyses were carried out using SAS version 9.4. Further details of statistical analyses are given as supplemental information.

Results

Overall, 303 study patients were eligible and randomized, allocating 151 patients toward clofarabine/PEG-ASP and 152 patients toward HIDAC/PEG-ASP (Figure 2, Table 1 and supplemental information). Of those patients, the main endpoint (i.e. MRD after randomized intervention) was reached by 296 patients, in close approximation to the planned sample size (n=295) (Table 2). There were no differences in patient characteristics regarding known risk factors other than a more frequent occurrence of

ETV6-RUNX1 in the clofarabine-treated cohort (Table 1). The incidence of hematopoietic stem cell transplantation (HSCT) in first complete remission due to persistent MRD was comparable between arms (n=11 vs n=12 HSCT in clofarabine and

HIDAC cohorts, respectively). T-ALL patients were similarly underrepresented in both randomized arms compared to the whole study cohort (5.3% (n=8) in the clofarabine and 5.9% (n=9) in the HIDAC cohort vs 14.2% (n=67) in the total cohort), mainly due to a greater proportion of T-ALL in the induction failure cohort (n=24 out of 31 (77%) patients) and in the HR-reduced cohort (15 out of 51 (29%) patients), both of which were excluded from randomization according to the study protocol (Table 2 and supplemental information).

MRD response

In the randomized treatment arms, we observed a rate of 44% MRD-positivity after high-

2 dose cytarabine vs 33% MRD-positivity after clofarabine in BCP-ALL (Pchi =0.03; left- sided Fisher test P=0.04). The overall reduction of MRD in BCP-ALL was significantly

more profound after clofarabine compared to cytarabine, with 93 clofarabine-treated patients vs 79 HIDAC-treated patients reaching MRD negativity, and a lower rate of patients with quantifiable MRD levels (six patients after clofarabine vs 18 patients after

HIDAC) (Cochran-Armitage trend test P=0.01; Table 2, Figure S1). This observation holds true in a sub-analysis of the patients with a higher burden of EOI MRD (≥10-3) who were stratified to the LR- or HR-intensified arms. Among those 73 patients, 27 patients were MRD-negative after clofarabine compared to 16 patients randomized to the HIDAC arm (Cochran-Armitage trend test P=0.02). In ETV6-RUNX1-rearranged ALL, which occurred more frequently in clofarabine-treated patients by chance, we observed an equivalent efficacy of the randomized nucleosides, reflecting a generally high sensitivity toward asparaginase in this prognostically favorable genetic subgroup of ALL (Table 1 and S3). To address a potential skewing effect of misbalanced ETV6-RUNX1 on the

MRD outcome of randomized groups, ETV6-RUNX1-negative ALL was analyzed separately, which confirmed greater activity of clofarabine compared to HIDAC

2 (Pchi =0.04210) (Table S3).

Importantly, after the randomized course in early consolidation (day 50 in BCP and day

64 in T-ALL patients), MRD maintained its prognostic relevance, with a significant impact on EFS and relapse rate in comparison to day 29 EOI MRD (Figure 3A,B).15 T-ALL patients of both randomized arms achieved comparable MRD reductions by day 64, although the number of T-ALL patients was very small (Table 1 and 2). Nevertheless, the test for trends in the overall cohort comprising both BCP- and T-ALL confirmed that

clofarabine was significantly more effective in MRD reduction compared to HIDAC

(Cochran-Armitage trend test P=0.01) (Table 2).

Outcome of randomized groups

No significant differences in outcome regarding event-free and overall survival were observed between the randomized arms (Figure 3C,D), with a median observation time of 3.7 years. There were also no significant differences in Cox regression analyses regarding the covariates gender, age (< vs ≥10 years), WBC (< vs ≥25/nl), ETV6-

RUNX1, and HSCT in first continuous remission as time-dependent variables. An additional stratified analysis confirmed that there were no significant differences in EFS or relapse rate between randomized courses according to the categories negative, positive n.q., and quantifiable MRD on day 50. Besides events that were anticipated upon quantifiable MRD after randomized intervention, several relapses occurred in

MRD-negative and -positive not-quantifiable patients in both randomized treatment arms, accounting for the observed lack of surrogacy of MRD in the outcome analysis

(Table S4). There was no evidence of a mutual impact between the randomizations at early consolidation and delayed intensification in this study, as shown by very similar pEFS in the latter randomized arms (log-rank test P=0.88 for patients receiving doxorubicin and log-rank test P=0.50 for patients receiving daunorubicin during delayed intensification).

Toxicity

No statistically significant differences in the incidence of severe or persistent toxicities between randomized treatment elements or in the subsequent treatment realization were documented (Figure 4, Table S2A,B). In particular, severe grade 3 or 4 skin toxicities were not observed in either treatment arm, but clofarabine was more frequently associated with grade 2 skin toxicities. With regard to hepatotoxicity, an elevation of transaminases (aspartate and alanine transaminases (AST and ALT, respectively)) was significantly more often reported after clofarabine than after HIDAC, and then spontaneously resolved without exception after each randomized treatment element before the start of subsequent chemotherapy. Accordingly, time intervals between the randomized courses and the subsequent treatment elements were similar, with a median of 22 days (range 20–38 days) after clofarabine/PEG-ASP and 19 days (range

18–38 days) after HIDAC/PEG-ASP. Incidence and degree of myelotoxicity differed slightly between clofarabine and HIDAC (Figure 4, Table S2A,B). Remarkably, when comparing CTC grades 0 to 2 against grades 3 and 4 for hemoglobin and platelets, clofarabine was associated with significantly less severe toxicities (Table S2B).

Clofarabine caused a more frequent grade 4 depletion of white blood cells suggesting a greater lymphotoxicity given that grade 4 reduction in neutrophil counts was comparable between randomized arms (Figure 4, Table S2A). Notwithstanding, the incidence of severe infections after randomized treatment was comparable (Figure 4, Table S2A,B).

Finally, the incidence of SAEs during the remaining treatment courses was very similar

(18 and 19 SAEs in the clofarabine vs HIDAC arm, respectively).

Discussion

As demonstrated in trial CoALL 08-09, clofarabine combined with PEG-asparaginase is effective in the eradication of minimal residual disease and well tolerated in the frontline treatment of ALL. In comparison to high-dose cytarabine/PEG-ASP, clofarabine/PEG-

ASP was superior in the overall reduction of an MRD burden. The frequency of MRD- positive BCP-ALL patients in the standard arm was lower than the predicted rate of 60%, likely due to a smaller sample size and the different distribution of risk strata in the preceding trial, CoALL 03-07.

Although the prognostic impact of MRD in BCP-ALL is still clearly discernible in early consolidation after the randomized courses of clofarabine vs HIDAC, the greater cytotoxic efficacy of clofarabine did not translate into an obvious improvement of outcome at the trial level after a median follow-up period of 3.7 years. This lack of surrogacy of MRD at early consolidation in a survival endpoint analysis could be explained by a small effect size, taking into account that only a single course of clofarabine was compared with high-dose cytarabine as a part of a complex multiagent chemotherapy backbone, the entirety of which determines treatment efficacy. Our trial design allowed for the detection of a ∼10% difference in outcome between randomized treatment arms at a power of 80%. Hence, the small sample size has to be considered with regard to the number of randomized patients required in order to perform a meaningful comparative analysis of survival in CoALL 08-09, which was a priori defined as a secondary objective in the study protocol.

Overall, clofarabine increased the rate of MRD negativity by 25% compared to HIDAC, which is an incremental improvement with borderline significance in contrast to a statistically more robust overall reduction of MRD after clofarabine (Table 2; Figure S1).

The occurrence of relapsing disease in MRD-negative patients after clofarabine (and

HIDAC) observed in this trial points at MRD as a time-dependent variable. In this regard, early achievement of MRD negativity at the end of induction is more predictive of outcome than achievement of MRD negativity later in treatment, most likely due to the emergence of resistant clones, i.e. MRD-negativity does not necessarily imply true eradication of the disease, but simply reflects a decrease to a level below the detection limit of the PCR-based MRD assay. Inversely, MRD positivity more reliably reflects outcome when measured later in treatment.15,16

In addition, the rarity of events after treatment of ALL in childhood might generally compromise surrogacy of MRD as a prognostic marker of outcome at the trial level. A previous multi-trial approach including 4830 patients with ALL demonstrated that EOI

MRD failed as a surrogate for treatment effects on EFS at the trial level, when dexamethasone and prednisone were compared in induction treatment of AIEOP-BFM

ALL and COG trials.17-19 This meta-analysis raised caution with regard to MRD as a surrogate marker for treatment decisions in randomized trials. In contrast to these trials, in which the stratifying decision was made after randomization, we can exclude that the evaluation of MRD after randomized intervention impacted a decision on the subsequent treatment in CoALL 08-09, since the ultimate stratification had been done before randomization on d29 in BCP-ALL and on d43 in T-ALL.

In this trial, we applied clofarabine at a dose of 40mg/m2 daily x 5 corresponding to the previously established single agent maximum-tolerated dose (MTD) in adult acute leukemia which is lower than the MTD of 52mg/m2 x 5 determined in pediatric patients with acute leukemia.2,20 The administration of high-dose clofarabine in conjunction with

PEG-asparaginase in early consolidation of CoALL 08-09 was feasible largely due to almost non-overlapping toxicities. By contrast, clofarabine given at a reduced dose level of 30mg/m2 x 5 or 20mg/m2 x 5, respectively, was associated with unacceptably severe infections and myelotoxicities in heavily pretreated pediatric patients with relapsed/refractory leukemia when combined with cyclophosphamide, etoposide, vincristine, and PEG-ASP in the COG trial AALL1131.7

Since MRD fell short as a surrogate marker in a true endpoint analysis of survival of randomized patient cohorts in CoALL 08-09, standard cytarabine treatment has not been replaced by clofarabine, despite its superior cytotoxic efficacy. Notwithstanding, given its favorable risk/benefit ratio, a further evaluation of clofarabine in combination with PEG-

ASP might be warranted as a second-line replacement or add-on strategy in specific patients, to reduce treatment-related morbidities or to augment the depth of molecular remission after antibody-based immunotherapy. 21,22 In particular, clofarabine/PEG-ASP could be tested in high-risk patients and compared with other established anti-leukemic agents that are burdened with severe acute and long-term toxicities, such as anthracyclines or the anti-metabolite methotrexate.23,24

Word count (main text): 2492

Data sharing

Individual patient data from the trial will not be shared publicly, since a data-sharing plan had not been included when ethical approval was requested. All original data can be obtained by the corresponding authors, please contact Dr. Gabriele Escherich: [email protected]

References

1 Xie KC, Plunkett W. Deoxynucleotide pool depletion and sustained inhibition of ribo-nucleotide reductase and DNA synthesis after treatment of human lymphoblastoid cells with 2-chloro-9-(2-deoxy-2-ﬂuoro-beta-D-arabinofuranosyl) adenine. Cancer Res.

1996;56(13):3030-3037.

2 Jeha S, Gandhi V, Chan KW, et al. Clofarabine, a novel nucleoside analog, is active in pediatric patients with advanced leukaemia. Blood. 2004;103(3):784-789.

3 Huang M, Inukai T, Miyake K, et al. Clofarabine exerts antileukemic activity against cytarabine-resistant B-cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression. Cancer Med. 2018;7(4):1297-1316.

4 Huguet F, Leguay T, Raffoux E, et al. Clofarabine for the treatment of adult acute lymphoid leukemia: the Group for Research on Adult Acute Lymphoblastic Leukemia intergroup. Leuk Lymphoma. 2015;56(4):847-857.

5 Wang H, Jones AK, Dvorak CC, et al. Population Pharmacokinetics of

Clofarabine as Part of Pretransplantation Conditioning in Pediatric Subjects before

Hematopoietic Cell Transplantation. Biol Blood Marrow Transplant. 2019;25(8):1603-

1610.

6 Hochberg J, Zahler S, Geyer MB, et al. The safety and efficacy of clofarabine in combination with high-dose cytarabine and total body irradiation myeloablative conditioning and allogeneic stem cell transplantation in children, adolescents, and young adults (CAYA) with poor-risk acute leukemia. Bone Marrow Transplant. 2019;54(2):226-

235.

7 Salzer WL, Burke MJ, Devidas M, et al. Toxicity associated with intensive postinduction therapy incorporating clofarabine in the very high-risk stratum of patients with newly diagnosed high-risk B-lymphoblastic leukemia: A report from the Children's

Oncology Group study AALL1131. Cancer. 2018;124(6):1150-1159.

8 Escherich G, zur Stadt U, Zimmermann M, Horstmann MA, CoALL study group.

Clofarabine in combination with pegylated asparaginase in the frontline treatment of childhood acute lymphoblastic leukaemia: a feasibility report from the CoALL 08-09 trial.

Br J Haematol. 2013;163(2):240-247.

9 Schramm F, Zimmermann M, Jorch N, et al. Daunorubicin during delayed intensification decreases the incidence of infectious complications - a randomized comparison in trial CoALL 08-09. Leuk Lymphoma. 2019;60(1):60-68.

10 Van der Velden VHJ, Cazzaniga G, Schrauder A, et al; European Study Group on

MRD detection in ALL (ESG-MRD-ALL). Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.

Leukemia. 2007;21(4):604-611.

11 Kaplan EL, Meier P. Nonparametric Estimation from Incomplete Observations. J

Am Stat Assoc. 1958;53(282):457-481.

12 Aalen OO. 1. The statistical analysis of failure time data (2nd edn). Kalbfleisch

JD, Prentice LR, Wiley-Interscience, Hoboken, New Jersey, 2002. Statistics in Medicine.

2004;23(21):3397-3398.

13 Gray RJ. A Class of K-Sample Tests for Comparing the Cumulative Incidence of a

Competing Risk. The Annals of Statistics. 1988;16(3):1141-1154.

14 Margolin BH. Test for Trend in Proportions. In: Kotz S, Johnson NL, Read CB, eds. Encyclopedia of Statistical Sciences. New York: John Wiley & Sons. 1988;vol.

9:334-336.

15 Borowitz MJ, Devidas M, Hunger SP, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study. Blood. 2008;111(12):5477-5485.

16 Brüggemann M, Kotrova M. Minimal residual disease in adult ALL: technical aspects and implications for correct clinical interpretation. Blood Adv. 2017;1(25):2456-

2466.

17 Galimberti S, Devidas M, Lucenti A, et al. Validation of Minimal Residual Disease as Surrogate Endpoint for Event-Free Survival in Childhood Acute Lymphoblastic

Leukemia. JNCI Cancer Spectr. 2018;2(4):pky069.

18 Möricke A, Zimmermann M, Valsecchi MG, et al. Dexamethasone vs prednisone in induction treatment of pediatric ALL: results of the randomized trial AIEOP-BFM ALL

2000. Blood. 2016;127(17):2101-2112.

19 Borowitz MJ, Wood BL, Devidas M, et al. Prognostic significance of minimal residual disease in high risk B-ALL: a report from Children's Oncology Group study

AALL0232. Blood. 2015;126(8):964-971.

20 Kantarjian HM , Gandhi V, Kozuch V, et al. Phase I clinical and pharmacology study of clofarabine in patients with solid and hematologic cancers. J Clin Oncol.

2003;21(6):1167-1173.

21 Locatelli F, Whitlock JA, Peters C, et al. Blinatumomab versus historical standard therapy in pediatric patients with relapsed/refractory Ph-negative B-cell precursor acute lymphoblastic leukemia. Leukemia. 2020;34(9):2473-2478.

22 Curren E, Stock W. Taking a “BiTE out of ALL”: blinatumomab approval for MRD- positive ALL. Blood. 2019;133(16):1715-1719.

23 Armenian S, Bhatia S. Predicting and Preventing Anthracycline-Related

Cardiotoxicity. Am Soc Clin Oncol Educ Book. 2018;38:3-12.

24 Bhojwani D, Sabin ND, Pei D, et al. Methotrexate-induced neurotoxicity and leukoencephalopathy in childhood acute lymphoblastic leukemia. J Clin Oncol.

2014;32(9):949-959.

Tables

Table 1: Demographics and clinical characteristics of randomized patients

High-dose Cytarabine Clofarabine (n=152) (n=151) No. (%) No. (%) P Value Immunophenotype B-precursor ALL 143 (94.1) 143 (94.7) .82 T-ALL 9 (5.9) 8 (5.3) Gender male 79 (52) 85 (56.3) .45 female 73 (48) 66 (43.7) Age at diagnosis < 10 years 123 (80.9) 119 (78.8) .65 ≥ 10 years 29 (19.1) 32 (21.2) WBC < 25/nl 101 (66.4) 110 (72.8) .73 ≥ 25/nl 51 (33.6) 41 (27.2) ETV6-RUNX1 rearrangement positive 30 (19.7) 47 (31.1) negative 117 (77) 104 (68.9) .02 unknown 5 (3.3) 0 (0) KMT2A rearrangement positive 2 (1.3) 2 (1.3) 1.0 negative 150 (98.7) 149 (98.7) Karyotype < 44 chromosomes 2 (1.3) 2 (1.3) 44-50 chromosomes 90 (59.2) 106 (70.2) .31 > 50 chromosomes 48 (31.6) 38 (25.2) unknown 12 (7.9) 5 (3.3) Treatment response BM day 15 M1 98 (64.5) 104 (68.9) M2 28 (18.4) 23 (15.2) .68 M3 4 (2.6) 5 (3.3) not available 22 (14.5) 19 (12.6) Risk Stratification Low-risk standard 57 (37.5) 62 (41.1) Low-risk intensified 20 (13.2) 19 (12.6)

High-risk standard 47 (30.9) 43 (28.5) High-risk intensified 28 (18.4) 27 (17.9)

Table 2 : MRD response toward clofarabine/PEG-ASP vs high-dose cytarabine/PEG-ASP

high-dose Clofarabine All Cytarabine No. (%) No. (%) P Value .03 Chi-square B-precursor ALL MRD d50 pos. 61 (44) 45 (33) 106 .04 Fisher MRD d50 neg. 79 (56.4) 93 (67.4) 172 .01 Cochran- B-precursor ALL MRD d50 pos. nq 43 (30.7) 39 (28.3) 82 Armitage MRD d50 ≥ 10-4 18 (12.9) 6 (4.3) 24 Trend Test .02 B-precursor ALL Cochran- MRD neg. 16 (21.9) 27 (37) 43 EOI MRD ≥10-3 Armitage Trend Test MRD d64 neg. 4 (44.4) 3 (37.5) 7 .94 Cochran- T-ALL MRD d64 pos. nq 3 (33.3) 4 (50.0) 7 Armitage MRD d64 ≥ 10-4 2 (22.2) 1 (12.5) 3 Trend Test

Figure legends:

Figure 1. Treatment overview.

A: Randomized treatment block clofarabine vs high-dose cytarabine, each combined with PEG-ASP.

B: Schematic overview of CoALL 08-09 protocol.

ADR, doxorubicin; BCP, B-cell precursor; BMP, bone marrow puncture; CNS, central nervous system; d, day; Dex, dexamethasone; DNR, daunorubicin ; EOI, end of induction; HIDAC, high-dose cytarabine; I, Induction; MRD, minimal residual disease;

PEG-ASP, pegylated asparaginase; R, randomization; VCR, vincristine;

Figure 2. Trial profile. Flow diagram according to CONSORT guidelines.

Figure 3. Outcome analyses in randomized patients.

A: Event-free survival (5 years of follow-up) in randomized patients according to MRD on day 50/64 after completion of randomized treatment courses. For comparative outcome probability analyses according to MRD levels, MRD negativity is denoted as 1, non- quantifiable (n.q.) MRD positivity is denoted as 2, and MRD ≥ 1 x 10-4 is denoted as 3.

B: Cumulative relapse rate (5 years of follow-up) in randomized B-precursor and T-ALL patients according to MRD on day 50/64.

C: Comparative analysis of overall survival (5 years of follow-up) in clofarabine/PEG-

ASP-treated vs HIDAC/PEG-ASP-treated ALL patients

D: Comparative event-free survival (pEFS) (5 years of follow-up) analysis in clofarabine/PEG-ASP-treated vs HIDAC/PEG-ASP-treated ALL patients

Figure 4:

Treatment-related toxicities in randomized patients according to treatment arm and CTC criteria

Supplementary Information:

 Supplemental Table 1:

Participating trial sites

 Supplemental Table 2:

Comparison of toxicities after clofarabine vs high-dose cytarabine (HIDAC) according to

CTC grades.

A: Comparison of all CTC grades

B: Comparison of CTC grades 0-2 vs 3 and 4.

Data are presented as No. (%).

 Supplemental Table 3:

MRD response in BCP-ALL patients according to their ETV6-RUNX1 status.

 Supplemental Table 4:

Distribution of relapses in the randomized arms according to the MRD level

 Supplemental Figure 1:

Comparison of MRD response in BCP-ALL

 CoALL 08-09 stratification algorithm

 Definition of event-free and overall survival

 Statistical analyses

 Study recruitment

Supplemental Table 1: Participating trial sites

Site City Country University Medical Center Ham- Hamburg Germany burg-Eppendorf, Clinic of Pediatric Hematology and Oncology Protestant Hospital of Bethel Bielefeld Germany Foundation, Department of Pediat- ric Hematology and Oncology University Hospital Bonn, Depart- Bonn Germany ment of Pediatric Hematology/On- cology Hospital Bremen-Mitte, Depart- Bremen Germany ment of Pediatric Hematology and Oncology Helios Hospital Krefeld, Depart- Krefeld Germany ment of Pediatric Hematology and Oncology University Medical Center of the Mainz Germany Johannes Gutenberg University Mainz, Department of Pediatric Hematology/Oncology University Hospital, Ludwig Maxi- Munich Germany milian Munich, Dr. von Hauner Children's Hospital Medical Faculty, Heinrich Heine Düssel- Germany University Düsseldorf, Pediatric dorf Oncology, Hematology and Clini- cal Immunology

Supplemental Table 2A,B : Comparison of toxicities

Table 2A:

high-dose Clofarabine, P Value Cytarabine, No. (%) (Fisher)

No. (%) Grade 0 13 (8.7) 15 (10.0) Grade 1 90 (60.0) 92 (61.3) Grade 2 37 (24.7) 40 (26.7) General condition .38493 Grade 3 8 (5.3) 3 (2.0) Grade 4 2 (1.3) 0 Total 150 150 Grade 0 1 (0.7) 1 (0.7) Grade 1 6 (4.0) 4 (2.6) Grade 2 33 (21.9) 56 (37.1) Hemoglobin .03460 Grade 3 87 (57.6) 75 (49.7) Grade 4 24 (15.9) 15 (9.9) Total 151 151 Grade 0 0 0 Grade 1 0 0 Grade 2 5 (3.3) 1 (0.7) WBC < .0001 Grade 3 60 (39.5) 8 (5.3) Grade 4 87 (57.2) 142 (94.0) Total 152 151 Grade 0 1 (0.8) 1 (0.8) Grade 1 0 1 (0.8) Grade 2 1 (0.8) 0 Neutrophils .37821 Grade 3 8 (6.1) 3 (2.3) Grade 4 122 (92.4) 123 (96.1) Total 132 128 Grade 0 1 (0.7) 15 (9.9) Grade 1 4 (2.6) 12 (7.9) Grade 2 9 (5.9) 17 (11.3) Platelets < .0001 Grade 3 87 (57.2) 83 (55.0) Grade 4 51 (33.6) 24 (15.9) Total 152 151 Grade 0 46 (30.7) 106 (70.7) Grade 1 84 (56.0) 34 (22.7) Number of platelet Grade 2 16 (10.7) 8 (5.3) < .0001 transfusions Grade 3 4 (2.7) 2 (1.3) Grade 4 0 0 Total 150 150 Grade 0 44 (29.1) 65 (43.0) Grade 1 11 (7.3) 12 (7.9) Grade 2 82 (54.3) 63 (41.7) Infections .07835 Grade 3 13 (8.6) 11 (7.3) Grade 4 1 (0.7) 0 Total 151 151 Grade 0 41 (27.2) 67 (44.4) Grade 1 65 (43.0) 55 (36.4) Fever .01048 Grade 2 41 (27.2) 28 (18.5) Grade 3 3 (2.0) 1 (0.7)

Grade 4 1 (0.7) 0 Total 151 151 Grade 0 44 (29.1) 70 (46.4) Grade 1 34 (22.5) 23 (15.2) Grade 2 61 (40.4) 48 (31.8) Days in hospital .03230 Grade 3 8 (5.3) 8 (5.3) Grade 4 4 (2.6) 2 (1.3) Total 151 151 Grade 0 122 (80.3) 123 (88.7) Grade 1 15 (9.9) 12 (8.0) Grade 2 12 (7.9) 3 (2.0) Stomatitis .09755 Grade 3 2 (1.3) 1 (0.7) Grade 4 1 (0.7) 1 (0.7) Total 152 150 Grade 0 132 (87.4) 125 (83.9) Grade 1 15 (9.9) 16 (10.7) Grade 2 4 (2.6) 6 (4.0) Diarrhea .54837 Grade 3 0 2 (1.3) Grade 4 0 0 Total 151 149 Grade 0 142 (93.4) 148 (98.0) Grade 1 9 (5.9) 3 (2.0) Grade 2 1 (0.7) 0 Creatinine .11123 Grade 3 0 0 Grade 4 0 0 Total 152 151 Grade 0 103 (68.2) 95 (63.3) Grade 1 32 (21.2) 33 (22.0) Grade 2 14 (9.3) 17 (11.3) Bilirubine .72543 Grade 3 2 (1.3) 4 (2.7) Grade 4 0 1 (0.7) Total 151 150 Grade 0 18 (12.1) 10 (6.7) Grade 1 50 (33.6) 41 (27.3) Grade 2 43 (28.9) 27 (18.0) Transaminases < .0001 Grade 3 36 (24.2) 52 (34.7) Grade 4 2 (1.3) 20 (13.3) Total 149 150 Grade 0 147 (96.7) 144 (96.6) Grade 1 4 (2.6) 4 (2.7) Peripheral neurotoxi- Grade 2 1 (0.7) 1 (0.7) 1.0000 city Grade 3 0 0 Grade 4 0 0 Total 152 149 Grade 0 149 (98.0) 150 (99.3) Grade 1 3 (2.0) 0 Grade 2 0 0 Central neurotoxicity .24752 Grade 3 0 0 Grade 4 0 1 (0.7) Total 152 151 Grade 0 135 (98.5) 133 (97.8) Arrhythmia Grade 1 0 2 (1.5) .62177 Grade 2 1 (0.7) 1 (0.7)

Grade 3 1 (0.7) 0 Grade 4 0 0 Total 137 136 Grade 0 85 (98.8) 76 (98.7) Grade 1 0 0 Grade 2 0 1 (1.3) Cardiac dysfunction .72317 Grade 3 1 (1.2) 0 Grade 4 0 0 Total 86 77 Grade 0 141 (92.8) 96 (64.4) Grade 1 6 (3.9) 33 (22.1) Grade 2 5 (3.3) 20 (13.4) Skin condition < .0001 Grade 3 0 0 Grade 4 0 0 Total 152 149 Grade 0 .. .. Grade 1 1 (100) .. Veno-occlusive dise- Grade 2 .. .. ase Grade 3 .. .. Grade 4 .. .. Total 1 0 Grade 0 151 (100) 149 (99.3) Grade 1 0 1 (0.7) Grade 2 0 0 Thrombosis .49834 Grade 3 0 0 Grade 4 0 0 Total 151 150

Supplemental Table 2B:

High-dose Clofarabine, P Value Cytarabine, No. (%) (Fisher) No. (%) Grade 0-2 140 (93.3) 147 (98.0) General condi- Grade 3/4 10 (6.7) 3 (2.0) .08529 tion Total 150 150 Grade 0-2 40 (26.5) 61 (40.4) Hemoglobin Grade 3/4 111 (73.5) 90 (59.6) .01451 Total 151 151 Grade 0-2 5 (3.3) 1 (0.7) WBC Grade 3/4 147 (96.7) 150 (99.3) .21409 Total 152 151 Grade 0-2 2 (1.5) 2 (1.6) Neutrophils Grade 3/4 130 (98.5) 126 (98.4) 1.0000 Total 132 128 Grade 0-2 14 (9.2) 44 (29.1) Platelets Grade 3/4 138 (90.8) 107 (70.9) < .0001 Total 152 151 Grade 0-2 146 (97.3) 148 (98.7) Number of plate- Grade 3/4 4 (2.7) 2 (1.3) .68433 let transfusions Total 150 150 Grade 0-2 137 (90.7) 140 (92.7) Infections Grade 3/4 14 (9.3) 11 (7.3) .67696 Total 151 151 Grade 0-2 147 (97.4) 150 (99.3) Fever Grade 3/4 4 (2.6) 1 (0.7) .37083 Total 151 151 Grade 0-2 139 (92.1) 141 (93.4) Days in hospital Grade 3/4 12 (7.9) 10 (6.6) .82534 Total 151 151 Grade 0-2 149 (98.0) 148 (98.7) Stomatitis Grade 3/4 3 (2.0) 2 (1.3) 1.0000 Total 152 150 Grade 0-2 151 (100) 147 (98.7) Diarrhea Grade 3/4 0 2 (1.3) .24584 Total 151 149 Grade 0-2 149 (98.7) 145 (96.7) Bilirubine Grade 3/4 2 (1.3) 5 (3.3) .28247 Total 151 150 Grade 0-2 111 (74.5) 78 (52.0) Transaminases Grade 3/4 38 (25.5) 72 (48.0) < .0001 Total 149 150 Grade 0-2 152 (100) 150 (99.3) Central neuroto- Grade 3/4 0 1 (0.7) .49835 xicity Total 152 151 Grade 0-2 136 (99.3) 136 (100) Arrhythmia Grade 3/4 1 (0.7) 0 1.0000 Total 137 136 Grade 0-2 85 (98.8) 77 (100) Cardiac dysfunc- Grade 3/4 1 (1.2) 0 1.0000 tion Total 86 77

Supplemental Table 3: MRD response in BCP-ALL patients according to their ETV6-RUNX1 status.

Table 3A: MRD response to clofarabine vs high-dose cytarabine in ETV6/RUNX1-negative BCP-ALL patients

high-dose p(chi)=.04210 Cytarabine Clofarabine All N % N % N MRD Day 50 MRD Day 50 neg. 54 50.0 58 62.4 112 MRD Day 50 pos. nq 37 34.3 30 32.3 67 MRD Day 50 pos. 17 15.7 5 5.4 22

Table 3B: MRD response to clofarabine vs high-dose cytarabine in ETV6/RUNX1-positive BCP-ALL patients

high-dose p(chi)=.94619 Cytarabine Clofarabine All N % N % N MRD Day 50 MRD Day 50 neg. 22 75.9 35 77.8 57 MRD Day 50 pos. nq 6 20.7 9 20.0 15 MRD Day 50 pos. 1 3.4 1 2.2 2

Supplemental Table 4: Distribution of relapses in absolute numbers in the randomized arms according to MRD level

Clofarabine (n) high-dose Cytarabine (n) MRD negative after clofarabine/ high-dose cytarabine 5 3 BCP-ALL 4 2 T-ALL 1 1 MRD positive n.q. after clofarabine/ high-dose cytarabine 8 5 BCP-ALL 7 5 T-ALL 1 0 MRD ≥10-4 after clofarabine/ high-dose cytarabine 2 6 BCP-ALL 2 5 T-ALL 0 1 Total 15 14

Supplemental Figure 1: Comparison of MRD response in BCP-ALL

Comparison of MRD response after clofarabine vs high-dose cytarabine, each combined with

PEG-ASP in BCP-ALL. MRD distribution at end of induction on day 29: clofarabine ≥ 1 x 10-2

4.6%, ≥ 1 x 10-3 26.5%, ≥ 1 x 10-4 24.5%; ≥ 1 x 10-6 43.7%; HIDAC ≥ 1 x 10-2 7.2%, ≥ 1 x 10-3

24.3%, ≥ 1 x 10-4 27% ≥ 1 x 10-6 41.4%; MRD distribution on day 50 after clofarabine: ≥ 1 x 10-2

0%, ≥ 1 x 10-3 2%, ≥ 1 x 10-4 2.6%, ≥ 1 x 10-6 28.5%, negative 64%; after HIDAC: ≥ 1 x 10-2 0%,

≥ 1 x 10-3 5.9% ≥ 1 x 10-4 7.2%, ≥ 1 x 10-6 30.3%, negative 54%.

CoALL 08-09 stratification algorithm

At diagnosis, patients were stratified according to conventional risk criteria, allocating patients aged ≥10 years, with a T- or pro-B cell immunophenotype or with a white blood cell count

(WBC) ≥25/nl to the high-risk (HR) arm, and all others to the low-risk (LR) arm. A second, more refined stratification was applied at EOI based on cytomorphological remission, molecular cytogenetics and in vivo MRD testing11. Patients not reaching remission at the end of induction, carrying a KMT2A-rearrangement or exhibiting a hypodiploid karyotype were also al- located to the HR arm. Ultimately, based on EOI MRD, three stratification arms were defined per risk group. B-precursor (BCP)-ALL patients with a negative MRD result and T-ALL patients with MRD EOI <10-3 were stratified to receive reduced treatment (LR- or HR-reduced), and were not eligible for randomization at consolidation and reinduction. BCP-ALL patients with

EOI MRD ≥10-3 and T-ALL patients with MRD ≥10-3 after the first course of consolidation were stratified to receive intensified treatment (LR- or HR-intensified). The remaining patients were assigned to standard treatment (LR- or HR-standard).

From 1 November 2013 to 31 December 2019, 476 protocol patients were enrolled in phase III of CoALL 08-09. All patients who were EOI MRD-negative (n=108) and patients with induction failure (n=31) were excluded from randomization, as were three patients who died during induction. 31 patients could not be randomized because of parental or patients’ refusal (n=14), technical non-feasibility (n=14), or severe adverse events (SAE) during induction (n=3).

Definition of event-free and overall survival

EFS was the time from diagnosis to the first event, defined as failure of protocol treatment

(non-remission: persistence of leukemic blasts ≥5% in the bone marrow (BM) until day 56 of treatment), induction death, relapse (re-emergence of blasts ≥25% or increasing blast counts in two consecutive BM biopsies after complete continuous remission and/or manifestation of

ALL in CNS and/or any extramedullary site by cytomorphology), death by any cause while in remission, secondary malignancy or censoring at last follow-up. OS was defined as the time

from diagnosis to death by any cause or censoring at last follow-up. Cox regression was used for multivariate analysis of the randomization groups taking into account known risk factors as covariates.

Statistical analyses

Sample size for randomization was calculated according to estimations of the primary endpoint, i.e. MRD of BCP-ALL after the first course of consolidation, based on the preceding trial,

COALL 07-03. We estimated that 60% of patients in the control group who were MRD-positive prior to intensification would exhibit a detectable MRD level after the administration of

HIDAC/PEG-ASP. We required 136 patients randomized to each group in order to demon- strate a 25% reduction yielding 45% MRD-positive patients with alpha=5% (one-sided) and beta=20%. Two interim analyses were planned in the study protocol, yielding a significance level of 0.042 in the final analysis.

For the randomized treatment element, local trial centers documented toxicities using a specific toxicity form based on NCI Common Toxicity Criteria, version 2.0. An additional field was implemented to capture the incidence and length of hospitalization. A lack of treatment-related hospitalization was defined as grade 0, <5 days:1, 5–10 days: 2, 10–15 days: 3, and >15 days of hospitalization corresponded to grade 4.

Study recruitment

From 1 November 2013 to 31 December 2019, 476 protocol patients were enrolled in phase III of CoALL 08-09. Among those, 108 patients achieved EOI MRD-negativity (n=108), 31 patients underwent an induction failure, and three patients died during induction who were not eligible for randomization. In addition, 31 patients could not be randomized because of parental or patients’ refusal (n=14), technical non-feasibility (n=14), or severe adverse events (SAE) during induction (n=3).