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Differing Outcomes of Patients with High Hyperdiploidy and ETV6-RUNX1 Rearrangement in Korean Pediatric Precursor B Cell Acute Lymphoblastic Leukemia

Purpose: Recent cooperative trials in pediatric acute lymphoblastic leukemia (ALL) report long-term event-free survival (EFS) of greater than 80%. In this study, we analyzed the outcome and prognostic factors for patients with precursor B cell ALL (n=405) diagnosed during a 10-year period (2005–2015) at our institution. Materials and Methods All patients were treated with a uniform institutional regimen based on four risk groups, except for steroid type; patients diagnosed up till 2008 receiving dexamethasone, while subsequent patients received prednisolone. None of the patients received cranial irradiation in first complete remission. Results The 10-year EFS and overall survival was 76.3%±2.3% and 85.1%±1.9%. Ten-year cumulative incidence of relapse, any central nervous system (CNS) relapse and isolated CNS relapse was 20.8%±2.2%, 3.7%±1.1%, and 2.5%±0.9%, respectively. A comparison of established, good prognosis genetic abnormalities showed that patients with high hyperdiploidy had significantly better EFS than those with ETV6-RUNX1 rearrangement (10-year EFS of 91.2%±3.0% vs. 79.5%±4.4%, p=0.033). For the overall cohort, male sex, infant ALL, initial CNS involvement, and Philadelphia chromosome (+) ALL were significant factors for lower EFS in multivariate study, while high hyperdiploidy conferred favorable outcome. For high and very high risk patients (n=231), high hyperdiploidy was the only significant factor for EFS in multivariate study. Conclusion Regarding good prognosis genetic abnormalities, patients with high hyperdiploidy had significantly better outcome than ETV6-RUNX1(+) patients. High hyperdiploidy was a major, favorable prognostic factor in the overall patient group, as well as the subgroup of patients with higher risk.


Introduction
Recent multi-center and multi-national cooperative trials in pediatric acute lymphoblastic leukemia (ALL) report long-term event-free survival (EFS) rates of > 80% and overall survival (OS) rates of ≥ 90% [1][2][3]. Regarding the overall treatment strategy for pediatric ALL, the addition of an intensification or reinduction phase for all patients regardless of risk group contributed to better outcome [4][5][6]. For central nervous system (CNS) prophylaxis, limiting cranial irradiation to high risk precursor B cell (Pre-B) and T cell ALL patients at a dose of 12 Gy was found to be a viable option to minimize long-term neurologic sequelae [7]. Other cooperative studies eliminated cranial irradiation in all patients while undertaking CNS prophylaxis through systemic and intrathecal chemotherapy, and showed low rates of CNS relapse [8,9].
Treatment according to risk group is a cardinal feature of contemporary pediatric ALL therapy, with the designation of risk group based on genetic features of the leukemic blast and response to early therapy, as measured by minimal residual disease [10,11]. Genetic abnormalities significantly influence outcome, with patients with high hyperdiploidy or ETV6-RUNX1 rearrangement known to have favorable prognosis [12].
Previously, we reported the outcome for 295 pediatric ALL patients treated with our institutional regimen, key features of which included intensification therapy based on risk, and the elimination of cranial irradiation in all patients in first complete remission (CR) [13].
Important prognostic variables in this study were high hyperdiploidy as a predictor of favorable outcome, while infant ALL had significantly worse survival. In this study, we undertook a follow-up analysis based on a larger number of patients than the original cohort, and limited to Pre-B ALL patients, to evaluate both rates of long-term survival, and significant predictors of outcome in Pre-B ALL. CANCER RESEARCH AND TREATMENT (CRT) 5 Korean Cancer Association This article is protected by copyright. All rights reserved.

Study group
Patients diagnosed with Pre-B ALL during a ten year period from January 2005 to June 2015 at the Department of Pediatrics, The Catholic University of Korea were included in the study (n=405). The study received institutional review board (IRB) approval (Study number KC19RESI0504). As the study was retrospective, the need for informed consent from the patients was waived by the IRB.

Risk group classification and treatment strategy
Key features of our risk group classification scheme and chemotherapy, which consisted of a uniform institutional regimen, were previously described [13]. Risk group classification was done after remission induction chemotherapy according to the following criteria for Pre-B ALL: • Low risk (all of the following criteria): age ≥ 1 and < 10 years old, initial white blood cell (WBC) count < 50 x 10 9 /L, trisomies of 4, 10 and 17 or ETV6-RUNX1 (+) • Standard risk: as for low risk, except for lack of trisomies of 4, 10 and 17 or ETV6-RUNX1 • High risk (any one of the following criteria, irrespective of low risk cytogenetic features): age ≥ 10 and < 15 years old, initial WBC count ≥ 50 x 10 9 /L and < 100 x 10 9 /L, initial CNS or testicular involvement, poor prephase steroid response, E2A-PBX1 (+), KMT2A rearrangement (+), minimal residual disease (MRD) (+) at end of remission induction (as measured by reverse transcription polymerase chain reaction (RT-PCR) or real-time quantitative (RQ)-PCR for patients with recurrent genetic abnormalities) • Very high risk (any one of the following, irrespective of low risk cytogenetic features): age ≥ 15 years old, initial WBC count ≥ 100 x 10 9 /L, Philadelphia chromosome-positive (Ph+) ALL, infant ALL, hypodiploidy (≤ 44 chromosomes), induction failure 6 Korean Cancer Association This article is protected by copyright. All rights reserved.
The major features of our treatment strategy included the following: • Four drug remission induction for all patients (vincristine, steroid, l-asparaginase, one dose of daunorubicin on day 1), preceded by 1 week of prephase steroid therapy (S1 Table).
• Intensification therapy based on patient risk, with low and standard risk patients receiving one phase of intensification, while high and very high risk patients received two phases of treatment (S2 Table -S5 Table).
• Maintenance therapy of 96 weeks for all patients regardless of gender. Treatment included pulses of vincristine and steroid every 4 weeks, as well as daily 6-mercaptopurine and weekly methotrexate (MTX). Of note, patients received high dose MTX (3g/m 2 ) and intrathecal MTX every 12 weeks during maintenance therapy (S6 Table).
• Omission of cranial irradiation in all patients. Treatment of CNS3 consisted of multiple triple intrathecal infusions (methotrexate, cytarabine, hydrocortisone) during remission induction, and use of dexamethasone in all phases of steroid treatment.
• Allogeneic hematopoietic cell transplantation (HCT) in first CR for patients with Ph+ ALL, hypodiploidy, and induction failure. Since September 2011, infant ALL with KMT2A rearrangement was also included as an indication for allogeneic HCT in first CR.
• For Ph+ ALL, imatinib was given concurrent with chemotherapy from Consolidation up till HCT, as reported previously [14]. Imatinib was not given after HCT, unless the patient showed an increase in RQ-PCR value for BCR-ABL1.
Patients diagnosed from 2005 -2008 received dexamethasone during all phases of steroid treatment, while patients diagnosed from 2009 onwards received prednisolone.

Study objectives
Primary objectives of the study were to determine the long-term EFS and OS of our study 7 Korean Cancer Association This article is protected by copyright. All rights reserved. group, as well as long-term incidence of overall relapse, CNS relapse, isolated CNS relapse, and nonrelapse mortality (NRM) for patients who achieved CR. We also aimed to identify key prognostic factors for EFS through both univariate and multivariate study for the entire study group, as well as for two subgroups based on risk (low and standard / high and very high).

Statistical analysis
EFS was determined from time of diagnosis to last follow-up in CR or first event, which included relapse, death, or primary refractory status. Patients who died during remission induction or due to primary refractory disease were considered to have events at time zero. OS was determined from date of diagnosis to last follow-up or death. Survival estimates were calculated by the Kaplan-Meier method, and univariate study of risk factors for EFS was done with the log-rank test. Multivariate analysis was done by Cox proportional hazard regression for variables found significant in univariate study. For patients who achieved CR, rate of relapse and NRM was determined by the cumulative incidence function. Patient follow-up was done up till June 30 th , 2019. P-value < 0.05 was considered significant.

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Prognostic factors
Patient gender, age at diagnosis, initial WBC count, CNS involvement at diagnosis, response to prephase steroid, and type of genetic abnormality of the leukemic blast proved to be significant predictors of EFS (Table 4). Also, patients treated with dexamethasone had significantly better EFS than those treated with prednisolone (10-year EFS 82.1±3.2% vs 70.3±4.3%, P=0.048). In multivariate study, male gender, infant ALL, initial CNS involvement, and Ph(+) ALL were significant factors for lower EFS, while high hyperdiploidy conferred favorable outcome ( Table 5).
Results of treatment according to patient subgroups were as follows.

1) Patient gender
The 10-year EFS for boys was significantly inferior to that for girls (71.7±3.1% vs. 83.4±3.0, p=0.016) ( Table 4). For the 52 boys who were initially treated with chemotherapy only in first CR and subsequently relapsed, the median time to relapse was 30.8 months from diagnosis (range: 3.5 -113.6 months). Twenty-one patients (40%) relapsed after treatment completion, 21 (40%) during maintenance therapy, while the remaining 10 patients (20%) relapsed early, prior to start of maintenance therapy.

2) Infant ALL
The 10-year EFS for the infants (n=21) was 42.9±10.8, significantly inferior to other age groups (Table 4). According to presence of KMT2A rearrangement, the 10-year EFS was 50.0±17.7% for the 8 patients without KMT2A rearrangement, none of whom received allogeneic HCT in first CR, and 38.5±13.5% for the 13 patients with KMT2A rearrangement (KMT2A (+)), 6 of whom received transplantation in first CR (p=0.708). Outcome for KMT2A (+) patients according to allogeneic HCT status was as follows: 10-year EFS of 50.0±20.4% Korean Cancer Association This article is protected by copyright. All rights reserved.

3) Initial CNS leukemia
Of the 6 patients with CNS leukemia at diagnosis, 4 experienced an event (10-year EFS 33.3±19.2%), including 2 patients with infection-related death during remission induction, 1 patient with BM relapse, and 1 patient with isolated CNS relapse.

4) Ph+ ALL
Twenty-five of the 31 patients with Ph+ ALL (81%) underwent allogeneic HCT in first CR; six patients did not undergo HCT due to lack of human leukocyte antigen fully-matched hematopoietic cell donor, early death, or comorbidities. Of the 25 patients who underwent first CR HCT, 4 patients relapsed and 6 patients died of NRM (10-year EFS 59.1±10.0%); of the 6 patients who did not receive HCT, 3 patients relapsed, and 2 patients died of NRM (10-year EFS 16.7±15.2%, p=0.057 when comparing the two groups). Ten-year OS of the Ph+ ALL subgroup was 65.7±8.9% (21/31).

Outcome and prognostic factors according to risk group
The proved significant in multivariate analysis.
Regarding the 231 patients with high and very high risk ALL, patient age at diagnosis, initial WBC count, CNS involvement, prephase steroid response, and the type of genetic abnormality had a significant impact on EFS in univariate study (S7 Table). In multivariate study, high hyperdiploidy was the only significant prognostic factor, with patients harboring this genetic abnormality having improved EFS (Hazard ratio 0.26, 95% confidence interval 0.08 -0.85, p=0.026).

Discussion
In this study, we report the outcome and prognostic factors of 405 pediatric Pre-B ALL patients diagnosed and treated with a uniform treatment regimen at our institution for a period of ten years. In multivariate study, we found that male gender, infant ALL, initial CNS Cranial irradiation has remained an important component of therapy for high risk ALL, either in the prophylactic setting, or for treatment of initial CNS involvement [3,11,21]. Recent cooperative trials, however, have shown that cranial irradiation may be replaced by intensive intrathecal chemotherapy, resulting in low incidence of CNS relapse [8][9][10]. In our study, none of the patients received cranial irradiation in first CR; the subsequent 10-year cumulative incidence of CNS relapse of 3.7% is similar to rates reported by recent trials in which cranial irradiation was incorporated as prophylaxis, or for the treatment of CNS involvement [3,11].
Only 6 patients in our study had CNS involvement at diagnosis (CNS3). Considering that 2 of the 4 events experienced by these patients were infection-related deaths during remission induction, rather than relapse, evidence from our data is limited regarding potential need for therapeutic cranial irradiation in this small subset of patients.    [2,8,9].
A clear limitation of our study was the lack of implementation of common MRD testing in our patients, with MRD done by RT-or RQ-PCR only for patients with recurrent genetic abnormalities, and the subsequent inability to confirm the well-known role of MRD as a prognostic factor. We also emphasize that due to our risk group stratification most of our patients were high or very high risk, in contrast to reported clinical trials in which most of the ALL patients were standard risk [6,8,10].