Identification of favorable subgroups of childhood high-hyperdiploid acute lymphoblastic leukemia in a modern therapy era – A retrospective international study of 3,231 patients

Identification of favorable subgroups of childhood high-hyperdiploid acute lymphoblastic leukemia in a modern therapy era – A retrospective international study of 3,231 patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification of favorable subgroups of childhood high-hyperdiploid acute lymphoblastic leukemia in a modern therapy era – A retrospective international study of 3,231 patients Andishe Attarbaschi, Amir Enshaei, Saskia Sonnenberg, Chloé Arfeuille, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8252282/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract B-cell acute lymphoblastic leukemia (B-ALL) with high-hyperdiploidy (HHD) represents the most prevalent genetic subtype of childhood ALL. This retrospective international study aimed to identify the HHD subgroup(s) having both the highest proportion of B-ALL cases and the best outcome. Data from 11,530 B-ALL patients, including 3,231 with HHD-B-ALL, treated between 1998 and 2018 in eight trials from seven international Berlin-Frankfurt-Münster Group members were analyzed. Eight HHD subgroups – including double and triple trisomies, trisomy 18, UK-good-risk (UK-GR) profile, and four different modal chromosome number (MCN) categories – were evaluated for 5-year event-free survival (EFS), overall survival, and relapse. Each subgroup was ranked by outcome measures and prevalence within the B-ALL trial cohorts. HHD frequency ranged from 19–35%. MCN 54–67 and UK-GR profile achieved the lowest overall ranking scores corresponding to the largest (up to 28% for MCN 54–67 and 26% for UK-GR among total B-ALL trial cohorts) and best HHD subgroups (EFS ranged from 80.5–92.8% for MCN 54–67 and 80.5–93.5% for UK-GR). Both an MCN of 54–67 and UK-GR profile define large, reproducible subgroups of HHD B-ALL with good prognosis. They represent strong candidates for risk-stratification of treatment de-intensification trials in the modern immunotherapy era. Health sciences/Diseases/Haematological diseases/Haematological cancer/Leukaemia/Acute lymphocytic leukaemia Health sciences/Risk factors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Modern risk-adapted treatment based on genetics and early response to therapy has yielded survival rates > 90% in childhood acute lymphoblastic leukemia (ALL) [ 1 , 2 ]. High-hyperdiploidy (HHD), mostly defined by a modal chromosome number (MCN) of 51–65 (67), defines the most prevalent ALL subentity and occurs in 25–30% of B-cell ALL (B-ALL) [ 3 – 5 ]. Recently, a more subtle classification scheme based on specific aneuploid forms has been proposed where classical HHD forms contain di-, tri, and tetrasomies while non-classical HHD contains di- and tetrasomies only [ 4 ]. Ploidy-related over-representation of chromosome 21 is the always-defining feature of HHD [ 3 , 4 ]. Traditionally, conventional G-banded karyotyping and/or the flow cytometry-based DNA index have been used for definition of HHD ALL with MCN peaks at 55 or 56 and a DNA index ≥ 1.16–1.6, respectively [ 6 – 13 ]. While HHD ALL exhibits evident biological, pharmacotypic and prognostic heterogeneity, overall outcome is good with an antimetabolite-based chemotherapy and event-free survival (EFS) rates of 80–85% [ 9 , 14 – 21 ]. However, there is no consensus among collaborative groups regarding the use of HHD as a whole group or its subgroups for risk stratification [ 6 , 7 , 11 , 14 – 16 , 22 – 24 ]. Whilst patients with HHD ALL have a relapse rate of about 10–15%, HHD ALL comprises 25% of all relapses [ 3 , 4 , 25 ]. No other genetic subgroup contributes more relapses, suggesting that improvement of prognosis of newly diagnosed HHD ALL is of clinical relevance. As several study groups have identified good-risk subsets among HHD ALL, the latter are ideal candidates for treatment de-escalation, in particular, nowadays, with less toxic immunotherapies changing the treatment landscape of pediatric B-ALL [ 14 – 16 , 22 , 26 , 27 ]. While most detailed long-term outcome data on HHD ALL come from Anglo-American studies, we aimed at establishing outcome measures of patients treated on European-based B-ALL protocols according to different definitions of low-risk/favorable HHD as assessed by standard G-banded karyotyping [ 11 , 14 – 16 ]. Importantly, the primary aim of the present study was not to compare outcome of different HHD profiles between the studies, but to identify the HHD profile having both the highest proportion of B-ALL cases and the best outcome within each study, followed by a combined analysis. Data collected via the international Berlin-Frankfurt-Münster (i-BFM) Study Group and presented herein, will serve as a reference benchmark for ongoing and future HHD ALL cohorts, defined by more modern technologies such as single nucleotide polymorphisms (SNP) arrays or optical genome mapping, and treated with the most contemporary therapy protocols [ 3 , 4 , 28 – 30 ]. PATIENTS AND METHODS Data on basic characteristics, treatment, and outcome of HHD ALL and non-HHD non-Down syndrome B-ALL patients, aged 1–18 years, included in trials where the recruitment period was between 1998 and 2018, were collected from seven study groups who were members of the i-BFM ALL Group (Data Supplement) [15, 22, 25, 31-38]. Patients were treated in one of nine trials (Data Supplement) [15, 22, 25, 31-38]. The median follow-up time for each trial but one (ALL-IC BFM 2009) exceeded 5 years. Of note, none but the EORTC-CLG (European Organisation for Research and Treatment of Cancer – Children’s Leukemia Group) 58951 trial used HHD for risk stratification, and for analysis of the Cooperative (CO) ALL Study Group patients, data of the COALL-07-03 and -08-09 trials were combined resulting in eight multicenter trials overall [22, 35, 36]. Moreover, the United Kingdom (UK)-good risk (GR) profile was developed using the UKALL97/99 and not the present UKALL2003 trial dataset [8]. A chromosome number of 51–67 defined HHD [3]. Diagnostic bone marrow and/or peripheral blood samples were analyzed by standard G-banded karyotyping. Fluorescence in-situ hybridization was acceptable to secure uncertain losses or gains of chromosomes, but should not have been the sole method for HHD-detection. Patients diagnosed only by DNA index, SNP arrays or other methodologies as well as patients with concomitant BCR::ABL1 , ETV6::RUNX1 , TCF3::PBX1 or KMT2A -involving fusions were excluded from the HHD B-ALL cohort. All karyotypes were curated to exclude masked haploidy/hypodiploidy from the HHD-cohort either by molecular testing or the pattern of tetrasomies [39]. Incomplete karyotypes, where only a chromosome count was possible or the analysis was regarded substandard, resulting in ≥3 chromosomes labelled as marker chromosomes, were also excluded. In addition, if only a range of the MCN was available, the highest value was used for final categorization. For analysis of the non-HHD B-ALL cases, only those with successful cytogenetic analysis (≥20 metaphases) were included. The following eight subgroups of HHD B-ALL were analysed: (1) triple trisomy (TT), defined by simultaneous trisomy of chromosomes 4, 10 and 17; (2) double trisomy (DT), defined by simultaneous trisomy of chromosomes 4 and 10; (3) trisomy 18 (T18); (4) UK-GR profile (simultaneous gain of chromosomes 17 and 18, or gain of chromosome 17 or 18, but no gain of chromosome 5 or 20; (5) MCN 51–53; (6) MCN 54–55; (7) MCN 54–67, and (8) MCN 56–67. Informed consent was obtained from the legal guardian(s) and/or patients, as appropriate. Trials were conducted according to the Declaration of Helsinki and with approval of the respective ethics committees. Statistical analysis Event-free survival was calculated from the date of diagnosis to the date of first event (relapse, second malignant neoplasm or death) and censored at the date of last follow-up for event-free patients. Overall survival (OS) was defined as the time from diagnosis to death from any cause or the date of last follow-up. Survival curves were estimated by the Kaplan-Meier method and compared by the log-rank test. Cumulative incidence of relapse (CIR) was calculated using cumulative incidence functions for competing events, constructed by the method of Kalbfleisch and Prentice and compared with Gray’s test. The Chi-squared test was used to compare proportions and the Kruskal-Wallis test to compare the equality of medians. All outcome rates are quoted at 5 years. In order to identify the HHD-profile with both the highest proportion and the best outcome, rankings for each HHD-profile were calculated. The sum of ranking performance of each profile for each endpoint (with 1 being the best and 8 the worst) was weighted by both the difference of survival compared to the ranked number one outcome and the proportion of the total and HHD B-ALL cohorts (Data Supplement): (*: best performing; Δ: difference; r: ranking; k1-3: 5-year EFS, OS, and CIR). Each individual study group performed all statistical analyses according to the pre-defined definitions ensuring consistency. While we did not centralize individual patient data for this study, summary statistics were collected for all eight trials using a standardized form (Data Supplement). P-values ≤0.05 were considered statistically relevant. RESULTS Overall, 11,530 B-ALL patients treated across eight trials from seven study groups were included in this analysis (Table 1). Sex ratio, median age, median leukocyte counts, and National Cancer Institute (NCI) risk distribution for the HHD (n = 3,231) and non-HHD B-ALL (n = 8,299) patients enrolled into the respective trials are shown in Table 1. The frequency of HHD among B-ALL cases varied significantly from 19% in ALL-IC BFM 2009 to 35% in EORTC-CLG 58951 (p < 0.001; Fig. 1A) [22, 34]. However, there was no statistically significant difference with respect to sex, age, leukocyte count or NCI risk among the HHD B-ALL cases across the eight trials (Fig. 2). Hence, the difference in frequency more likely reflects differential cytogenetic success rates rather than genuine biological differences. Initially, we compared the outcome of B-ALL patients with and without HHD per trial. In six of eight trials, HHD patients had a lower 5-year CIR compared to other B-ALL patients, but the difference was significant in only three trials (Fig. 1B). In the remaining two trials – ALL-BFM-Austria (A) 2000 and Dutch Childhood Oncology Group (DCOG) ALL10 – HHD patients had a non-significant higher CIR compared to other B-ALL patients [32, 33]. The same pattern was observed for 5-year EFS and OS rates, where four trials reported significantly superior rates, two trials non-significant higher rates and two trials (ALL BFM-A 2000, DCOG ALL10) slightly lower rates for HHD patients (Fig. 1C/1D) [32, 33]. Several studies have proposed subgroups of HHD ALL, based on the pattern of specific chromosome gains or MCN, which have superior outcomes [9, 11, 14–16, 26, 40, 41]. Hence, we classified all 3,231 patients with HHD B-ALL into eight subgroups using consistent definitions based on cytogenetic karyotypes. The frequency of DT, TT, T18 and UK-GR profile varied between each trial (Fig. 3A). However, the pattern of variation was consistent for each subgroup ( i.e. , the same trial had the lowest frequency of each subgroup), suggesting that the differences were driven by the quality of the cytogenetic data ( i.e. , number of marker chromosomes) rather than by genuine biological heterogeneity. Consistent with this explanation, the frequency of the MCN subgroups (51–53, 54–55, and 56–67) was much more comparable across the trial datasets (Fig. 3B). To assess the potential clinical utility of each subgroup of HHD, we considered two key metrics: (1) the 5-year outcome measures, and (2) the proportion of B-ALL patients captured (Table 1). We opted for the proportion of B-ALL rather than the proportion of HHD B-ALL due to the variability in HHD-frequency across the trial datasets (Fig. 1A). To visualize the two metrics for all eight definitions across all eight trials, we plotted the 5-year outcome measures against the B-ALL proportion for 5-year CIR, EFS and OS (Fig. 4). In each plot, there is evidence of clustering, with the UK-GR profile, T18 and MCN 54–67 subgroups tending to have the highest frequency and best outcome. Intriguingly, the plots also showed the subgroup of MCN 51–53 to have the lowest frequency and worst outcome. To compare each subgroup within each trial, we developed a ranking system, which considers the difference in outcome for each endpoint as well as the size of the putative subgroup using both the total B-ALL and HHD B-ALL cohorts as reference points (Fig. 5A). Using this system, all eight subgroups were ranked from one to eight for each trial separately (Fig. 5B). As a next step, we summed the ranks across the eight trials so that each subgroup was assigned an overall score of between eight (always ranked first) to 64 (always ranked last). As a result, the two highest-ranking subgroups were MCN 54–67 and UK-GR profile with overall scores of 16 and 17, respectively (Fig. 5C). Patients with MCN 51–53 had the worst outcome in all trials and, hence, achieved an overall score of 64. The remaining subgroups had overall scores of between 28 and 51. Accordingly, apart from MCN 54–67 (ranked #1 in 5 trials) and UK-GR profile (ranked #1 in 2 trials), only T18 reached rank #1 (overall score: 28) in one trial (UKALL2003). Moreover, the second worst subgroup was MCN 54–55 (ranked #7 in 5 trials) with an overall score of 51. To further explore the UK-GR profile and MCN 54–67, we evaluated outcomes among patients enrolled on the eight trials who were classified in either one, both, or neither subgroup. Among 712 UKALL2003 HHD B-ALL patients, the majority (n = 493; 69%) were positive for both UK-GR and MCN 54–67, with a 5-year EFS of 93.9% (95% CI, 91.3–95.7). Patients with UK-GR profile only (n = 75) had a 5-year EFS of 90.7% (95% CI, 81.4–95.4), whereas those with MCN 54–67 profile only (n = 93) had a 5-year EFS of 83.6% (95% CI, 74.3–89.8). Patients belonging to neither subgroup (n = 51) had a 5-year EFS of 86.2% (95% CI, 73.2–93.2). An analogous analysis was performed for the 843 HHD B-ALL patients from trial AIEOP-BFM ALL 2009. The corresponding 5-year EFS rates were 91.3% (95% CI, 88.7–93.3), 89.1% (95% CI, 80.7–94.0), 91.9% (95% CI, 84.5–95.9), and 73.1% (95% CI, 57.5–83.7) for patients positive for both UK-GR and MCN 54–67 (n = 603, 72%), UK-GR only (n = 93), MCN 54–67 only (n = 102), and neither subgroup (n = 45), respectively. Analyses of the other six trials, each comprising fewer HHD patients than UKALL2003 and AIEOP-BFM ALL 2009, revealed that – with the exception of ALL-IC BFM 2009 – the vast majority of HHD cases were positive for both the UK-GR and MCN 54–67 profiles. The latter consistently demonstrated the most favorable EFS rates. In contrast, outcomes for the remaining three subgroups showed no consistent pattern across the six trials (Data Supplement). DISCUSSION The present i-BFM ALL study reports on a large cohort of 3,231 children and adolescents with HHD ALL out of 11,530 patients with B-ALL. These HHD cases were consistently classified into eight potential low-risk subgroups, defined by either specific chromosomal gains or distinct categories of MCN [ 14 , 15 ]. Within this multinational study framework, we clearly demonstrated that both an MCN of 54–67 and the UK-GR profile represent robust genetic markers identifying the largest HHD subgroups (up to 28% for MCN 54–67 and 26% for UK-GR) with the most favorable outcomes among total B-ALL cases. Five-year EFS ranged from 80.5–92.8% for MCN 54–67 and from 80.5–93.5% for UK-GR profile across our trial datasets. Importantly, both subsets are easy to detect in clinical trial settings – regardless of whether conventional G-banded karyotyping or more advanced methodologies are employed [ 14 , 15 , 29 ]. While the MCN requires only assessment of the total chromosome count, the UK-GR profile is based on assessment of four specific chromosomes [ 14 , 15 ]. These findings could be particularly relevant for resource-limited settings, where conventional cytogenetics (or DNA index), sometimes complemented by fluorescence in-situ hybridization, remains the standard diagnostic approach for HHD [ 12 , 13 , 42 ]. The data collected from the individual trials revealed a substantial variation in the frequency of HHD among their total B-ALL cohorts, ranging from 19% to 35%. As there were no relevant differences with regard to basic demographics such as sex, age or leukocyte counts at diagnosis, this variability confirms that conventional G-banded karyotyping has inherent limitations overlooking HHD cases [ 30 , 43 ]. Therefore, implementation of newer technologies, such as SNP arrays or next-generation-sequencing-based approaches may improve the accurate detection of HHD and its clinically relevant subsets in the future [ 30 ]. With the emergence of less toxic immunotherapies such as blinatumomab, next-generation pediatric low (standard)-risk ALL trials aim to achieve substantial treatment de-escalation while maintaining high cure rates of 90–95% in this subset [ 1 , 29 ]. Previous studies have primarily focused on identifying intermediate- and high-risk genetic abnormalities ( i.e. , IKZF1 plus , IKZF1 -deletion, hypodiploidy, iAMP21, TCF3 - or KMT2A -rearrangements) to guide therapy intensification and improve outcomes [ 29 , 31 , 44 – 49 ]. In contrast, good-risk genetics such as ETV6::RUNX1 or HHD have, at least in some European-based protocols, been neglected in recent decades for risk stratification and been utilized to support treatment de-intensification in only a few studies [ 3 , 31 , 32 , 34 – 38 , 50 , 51 ]. Interestingly, HHD was used to define the very low-risk group in our included EORTC-CLG 58951 trial and, yet, retained a significant favorable prognostic impact (MCN 54–67 ranked #1) [ 22 ]. To address the objectives of the present study – namely, to identify the HHD subgroup with both the highest proportion of B-ALL patients and the most favorable outcome – we implemented a standardized approach across participating study groups. Each group was asked to provide not only general cohort characteristics, but also fully analyzed outcome data stratified according to the predefined eight HHD B-ALL subgroups, as compared with their B-ALL counterparts. This approach avoided collection of individual patient-level data from > 11,500 patients, while allowing simultaneous evaluation of CIR, EFS and OS for all eight subgroups and their relative frequencies among total B-ALL cases. Ranking across the eight trials identified MCN of 54–67 and UK-GR profile as the largest subgroups with the best prognoses, as reflected by the lowest cumulative scores (16 and 17, respectively). Conversely, the MCN 51–53 subgroup exhibited the highest score possible (64), thus, representing the smallest subgroup (3–7%) with the poorest outcomes (EFS ranged from 68.7–88.9%). A recent study by the St. Jude Children’s Research Hospital (SJCRH) conducted a comparable analysis of 915 B-ALL patients enrolled in two consecutive frontline trials, using six different HHD-definitions: (1) MCN 51–67, (2) DNA index 1.16–1.6 (with ≥ 1.16 corresponding to MCN ≥ 54 and 1.6 to approximately 74 chromosomes), (3) UK-GR profile, (4) T18, (5) DT, and (6) TT [ 16 ]. In multivariable analysis, only the DNA index of 1.16–1.6 – comprising 25.3% of all patients – remained as an independent prognostic marker for CIR and EFS [ 16 ]. These findings suggested that the DNA index of 1.16–1.6 might represent the most clinically practical HHD-definition in the context of SJCRH-based therapies, as it identifies a sufficiently large subgroup with excellent outcomes and combines favorable prognostic power with low cost, technical simplicity, and rapid turnaround. The main difference between the SJCRH and our analysis lies in methodology: we did not analyze HHD according to DNA index, whereas the SJCRH study did not perform separate analyses according to our MCN subgroups [ 16 ]. Specifically, the SJCRH study’s inclusion of patients with 51–53 chromosomes within the broader MCN 51–67 group likely diluted outcome measures, as these lower-MCN cases are associated with inferior prognosis (at least in our dataset). On the top of their prognostic factor analysis, the SJCRH study nicely demonstrated that ex-vivo drug sensitivities are associated with gain of specific chromosomes ( i.e. , trisomy 17 was significantly associated with L-asparaginase and 6-mercaptopurine sensitivity) [ 16 ]. Evident limitations of our multinational study include the retrospective nature of data collection over a 20-year period also encompassing trials initiated before 2000 (ALL-BFM-A 2000, EORTC-CLG 58951) [ 22 , 33 ]. Some of our participating groups may have missed cases of HHD B-ALL due to failed karyotyping or suboptimal cytogenetic quality. Moreover, chemotherapy protocols and risk stratification criteria varied among the participating trials. In conclusion, an MCN of 54–67 and the UK-GR profile define large subgroups of HHD B-ALL with very favorable prognosis. Both can serve as practical genetic markers to prospectively stratify children with B-ALL into low-risk trials, where substitution of conventional chemotherapy with immunotherapy and systematic reduction or modification of chemotherapeutic components can be evaluated in randomized settings [ 27 ]. Beyond genetic classification, early treatment response as assessed by minimal residual disease (MRD) remains the most critical predictor of relapse in childhood ALL [ 25 , 29 , 52 ]. Accordingly, the interplay between HHD and treatment response is planned for assessment in the upcoming AIEOP-BFM ALL 2025 trial for standard-risk B-ALLs defined by MCN 54–67 and MRD negativity at end-of-induction [ 23 ]. In contrast, patients with an MCN of 51–53 appear to form a numerically related, yet biologically distinct form of HHD, and, thus, not to be ideal candidates for treatment de-intensification but requiring further molecular characterization to elucidate the biological mechanisms underlying their inferior outcomes. Declarations DATA SUPPLEMENT Authors retain all rights in any data supplements associated with their articles. DATA AVAILABILITY The data supporting the findings of this study are available from the corresponding author upon request to AA. FUNDING AA and KN were supported by St. Anna Children’s Cancer Research Institute; SS, GC, AM, MS, and MZ were supported by Deutsche Krebshilfe e.V. (funding number 108588) for the AIEOP-BFM ALL 2009 trial; MC was supported by Chilean National Pediatric Cancer Program; RP and MLB received core funding from KiKa and JMB was supported by KiKa grant 264; LC's fellowship at the EORTC Headquarters was supported by the EORTC Cancer Research Fund (ECRF); CK and EZ were supported by NKFIH-OTKA K 143021 [Foundation for Children with Leukemia (Leukemias Gyermekekert Alapitvany)]. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST AA: Honoraria: Jazz Pharmaceuticals, Amgen, Novartis; Consulting or Advisory Role: Jazz Pharmaceuticals, Amgen, Novartis, Takeda Science Foundation; Travel, Accommodations, Expenses: Jazz Pharmaceuticals SS: Consulting and Advisory Role: SERB Pharmaceuticals, Jazz Pharmaceuticals GC: Research support: Amgen, Clinigen, Jazz Pharmaceuticals, Servier; Travel, Honoraria: Amgen, Clinigen, Jazz Pharmaceuticals SE: Consulting or Advisory Role: Jazz Pharmaceuticals AM: Travel: Jazz Pharmaceuticals; Consulting or Advisory Role: Jazz Pharmaceuticals MS: Research support: Shire, Clinigen, Jazz Pharmaceuticals, Servier, Amgen; Honoraria: Servier, Amgen, Clinigen, Jazz Pharmaceuticals All other authors declare no conflicts of interest. AUTHOR CONTRIBUTIONS Conception and design: AA, AE, AVM and MZ. Provision of study materials or patients: AA, CA, DB, LB, NB, JMB, MC, GC, HC, LC, SE, GE, HAG-K, MLB, CK, JL, AM, HM, KN, RP, PR, MS, AV, EZ, JZ, AB, MZ and AVM. Collection and assembly of data: AA, CA, DB, LB, NB, JMB, MC, GC, HC, LC, SE, GE, HAG-K, MLB, CK, JL, AM, HM, KN, RP, PR, MS, AV, EZ, JZ, AB, MZ and AVM. Data analysis and interpretation: AA, AE, MZ, HAG-K, LC, and AVM. Manuscript writing: All authors. Final approval of manuscript: All authors. Accountable for all aspects of the work: All authors. ACKNOWLEDGEMENTS We thank all participating institutions, geneticists, biologists, and physicians for their support of the study. 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IKZF1(plus) Defines a New Minimal Residual Disease-Dependent Very-Poor Prognostic Profile in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia. J Clin Oncol. 2018;36:1240-1249. Moorman AV, Richards SM, Robinson HM, Strefford JC, Gibson BE, Kinsey SE, et al. Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21). Blood. 2007;109:2327-2330. Domenech C, Kicinski M, De Moerloose B, Piette C, Chahla WA, Kornreich L, et al. Results of the prospective EORTC Children Leukemia Group study 58081 in precursor B- and T-cell acute lymphoblastic leukemia. Hemasphere. 2024;8:e70025. Gottschalk H, Moricke A, Conter V, Schrappe M, Stary J, Cario G, et al. Reducing Daunorubicin in Induction Therapy in Children With B-Lineage ALL With Favorable Prognosis: Results of Phase III Trial AIEOP-BFM ALL 2009. J Clin Oncol. 2025;JCO2501357. O'Connor D, Enshaei A, Bartram J, Hancock J, Harrison CJ, Hough R, et al. Genotype-Specific Minimal Residual Disease Interpretation Improves Stratification in Pediatric Acute Lymphoblastic Leukemia. J Clin Oncol. 2018;36:34-43. Tables Table 1 is available in the Supplementary Files section. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files HHDALLTable1LEUKEMIAAA01122025V1.0.docx Table 1 HHDALLDataSupplementLeukemiaAA22112025V1.0.pdf Data Supplement Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":182485,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency and outcome of patients with high-hyperdiploid B-ALL per trial. (A) Frequency of high-hyperdiploid (HeH) among B-ALL patients per trial; (B-D) Comparison of 5-year cumulative risk of relapse (B), event-free survival (C) and overall survival (D) between high-hyperdiploid and other B-ALL patients per trial. Vertical bars indicate the 95% confidence interval.\u003cstrong\u003e \u003c/strong\u003eP-values were obtained using the log-rank test.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-8252282/v1/d9dfb8ebb29550ae0843d448.png"},{"id":98432242,"identity":"9446d73b-88a8-406d-af54-00f5e8201e49","added_by":"auto","created_at":"2025-12-17 16:49:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126944,"visible":true,"origin":"","legend":"\u003cp\u003eDemographic and clinical features of patients with high-hyperdiploid B-ALL per trial. Abbreviations: WBC, white blood cell count; NCI, National Cancer Institute; SR, standard risk; HR, high risk; * in 3 patients (2%) no NCI risk group was reported.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-8252282/v1/2e7ebb877105fa8719190a8a.png"},{"id":98218597,"identity":"b29f49dc-8ba7-4ead-b2ea-58d1f4fdf65d","added_by":"auto","created_at":"2025-12-15 11:03:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74633,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency of high-hyperdiploid B-ALL subgroups per trial. (A) Proportion of high-hyperdiploid cases with UKALL-good risk profile (UK-GR), trisomy 18 (T18), triple trisomy (TT: +4, +10, +17) and double trisomy (DT: +4, +10) for each trial; (B) Proportion of high-hyperdiploid cases with a modal number of 51–53 chromosomes, 54 – 55 chromosomes and 56–67 chromosomes.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-8252282/v1/da814b08d796631528263825.png"},{"id":98433928,"identity":"0c1124d4-021d-49b1-8397-97c58147de74","added_by":"auto","created_at":"2025-12-17 16:51:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":131376,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of the clinical utility of each high-hyperdiploid subgroup in B-ALL. Each plot shows the proportion of B-ALL cases captured by each subgroup (Y- axis) and the 5-year cumulative incidence of relapse (A), event free survival (B) and overall survival (C) rates (X-axis). In each plot, the colour represents different high- hyperdiploid subgroups and numbers indicate the different trials.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-8252282/v1/3562595810ad570c3854c499.png"},{"id":98218603,"identity":"36f76a90-2bf3-42c0-a39b-4cd438f8283f","added_by":"auto","created_at":"2025-12-15 11:03:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105656,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the outcome of each high-hyperdiploid (HeH) subgroup within each trial using a ranking system. (A) Schematic showing how the rankings were calculated for each subgroup; (B) Heatmap illustrating the rankings of each subgroup per trial. Dark blue indicates best performing subgroups in terms of outcome and size whereas red indicates the worst performing subgroups; (C) Bar chart evaluating the performance of each subgroup across all trials. The height of each bar indicates the sum of all rankings across all trials; so lower is better. The width of each bar represents the size of each subgroup.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-8252282/v1/6581b1ec19c35e0a3c76f0e1.png"},{"id":100371714,"identity":"baa71afe-c4b3-4454-bc29-8959c31a5b2d","added_by":"auto","created_at":"2026-01-16 08:10:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1242680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8252282/v1/b13c3f62-db65-4733-9e5a-2f379a709512.pdf"},{"id":98433810,"identity":"113aefab-3516-4b6e-92f1-717e547a61fc","added_by":"auto","created_at":"2025-12-17 16:51:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":46057,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"HHDALLTable1LEUKEMIAAA01122025V1.0.docx","url":"https://assets-eu.researchsquare.com/files/rs-8252282/v1/00467fea2bbfb7d772942611.docx"},{"id":98218601,"identity":"b5fe3958-4581-4237-92eb-558ec5a1eecf","added_by":"auto","created_at":"2025-12-15 11:03:06","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":755370,"visible":true,"origin":"","legend":"Data Supplement","description":"","filename":"HHDALLDataSupplementLeukemiaAA22112025V1.0.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8252282/v1/4046d154ca8a7072a2ed513b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Identification of favorable subgroups of childhood high-hyperdiploid acute lymphoblastic leukemia in a modern therapy era – A retrospective international study of 3,231 patients","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eModern risk-adapted treatment based on genetics and early response to therapy has yielded survival rates\u0026thinsp;\u0026gt;\u0026thinsp;90% in childhood acute lymphoblastic leukemia (ALL) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. High-hyperdiploidy (HHD), mostly defined by a modal chromosome number (MCN) of 51\u0026ndash;65 (67), defines the most prevalent ALL subentity and occurs in 25\u0026ndash;30% of B-cell ALL (B-ALL) [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recently, a more subtle classification scheme based on specific aneuploid forms has been proposed where classical HHD forms contain di-, tri, and tetrasomies while non-classical HHD contains di- and tetrasomies only [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Ploidy-related over-representation of chromosome 21 is the always-defining feature of HHD [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Traditionally, conventional G-banded karyotyping and/or the flow cytometry-based DNA index have been used for definition of HHD ALL with MCN peaks at 55 or 56 and a DNA index\u0026thinsp;\u0026ge;\u0026thinsp;1.16\u0026ndash;1.6, respectively [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While HHD ALL exhibits evident biological, pharmacotypic and prognostic heterogeneity, overall outcome is good with an antimetabolite-based chemotherapy and event-free survival (EFS) rates of 80\u0026ndash;85% [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, there is no consensus among collaborative groups regarding the use of HHD as a whole group or its subgroups for risk stratification [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhilst patients with HHD ALL have a relapse rate of about 10\u0026ndash;15%, HHD ALL comprises 25% of all relapses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. No other genetic subgroup contributes more relapses, suggesting that improvement of prognosis of newly diagnosed HHD ALL is of clinical relevance. As several study groups have identified good-risk subsets among HHD ALL, the latter are ideal candidates for treatment de-escalation, in particular, nowadays, with less toxic immunotherapies changing the treatment landscape of pediatric B-ALL [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile most detailed long-term outcome data on HHD ALL come from Anglo-American studies, we aimed at establishing outcome measures of patients treated on European-based B-ALL protocols according to different definitions of low-risk/favorable HHD as assessed by standard G-banded karyotyping [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Importantly, the primary aim of the present study was not to compare outcome of different HHD profiles between the studies, but to identify the HHD profile having both the highest proportion of B-ALL cases and the best outcome within each study, followed by a combined analysis.\u003c/p\u003e\u003cp\u003eData collected via the international Berlin-Frankfurt-M\u0026uuml;nster (i-BFM) Study Group and presented herein, will serve as a reference benchmark for ongoing and future HHD ALL cohorts, defined by more modern technologies such as single nucleotide polymorphisms (SNP) arrays or optical genome mapping, and treated with the most contemporary therapy protocols [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e"},{"header":"PATIENTS AND METHODS","content":"\u003cp\u003eData on basic characteristics, treatment, and outcome of HHD ALL and non-HHD non-Down syndrome B-ALL patients, aged 1–18 years, included in trials where the recruitment period was between 1998 and 2018, were collected from seven study groups who were members of the i-BFM ALL Group (Data Supplement) [15, 22, 25, 31-38]. Patients were treated in one of nine trials (Data Supplement) [15, 22, 25, 31-38]. The median follow-up time for each trial but one (ALL-IC BFM 2009) exceeded 5 years. Of note, none but the EORTC-CLG (European Organisation for Research and Treatment of Cancer – Children’s Leukemia Group) 58951 trial used HHD for risk stratification, and for analysis of the Cooperative (CO) ALL Study Group patients, data of the COALL-07-03 and -08-09 trials were combined resulting in eight multicenter trials overall [22, 35, 36]. \u0026nbsp;Moreover, the United Kingdom (UK)-good risk (GR) profile was developed using the UKALL97/99 and not the present UKALL2003 trial dataset [8].\u003c/p\u003e\n\u003cp\u003eA chromosome number of 51–67 defined HHD [3]. Diagnostic bone marrow and/or peripheral blood samples were analyzed by standard G-banded karyotyping. Fluorescence \u003cem\u003ein-situ\u003c/em\u003e hybridization was acceptable to secure uncertain losses or gains of chromosomes, but should not have been the sole method for HHD-detection. Patients diagnosed only by DNA index, SNP arrays or other methodologies as well as patients with concomitant \u003cem\u003eBCR::ABL1\u003c/em\u003e, \u003cem\u003eETV6::RUNX1\u003c/em\u003e, \u003cem\u003eTCF3::PBX1\u003c/em\u003e or \u003cem\u003eKMT2A\u003c/em\u003e-involving fusions were excluded from the HHD B-ALL cohort. All karyotypes were curated to exclude masked haploidy/hypodiploidy from the HHD-cohort either by molecular testing or the pattern of tetrasomies [39]. Incomplete karyotypes, where only a chromosome count was possible or the analysis was regarded substandard, resulting in ≥3 chromosomes labelled as marker chromosomes, were also excluded. In addition, if only a range of the MCN was available, the highest value was used for final categorization. For analysis of the non-HHD B-ALL cases, only those with successful cytogenetic analysis (≥20 metaphases) were included.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe following eight subgroups of HHD B-ALL were analysed: (1) triple trisomy (TT), defined by simultaneous trisomy of chromosomes 4, 10 and 17; (2) double trisomy (DT), defined by simultaneous trisomy of chromosomes 4 and 10; (3) trisomy 18 (T18); (4) UK-GR profile (simultaneous gain of chromosomes 17 and 18, or gain of chromosome 17 or 18, but no gain of chromosome 5 or 20; (5) MCN 51–53; (6) MCN 54–55; (7) MCN 54–67, and (8) MCN 56–67.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from the legal guardian(s) and/or patients, as appropriate. Trials were conducted according to the Declaration of Helsinki and with approval of the respective ethics committees. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvent-free survival was calculated from the date of diagnosis to the date of first event (relapse, second malignant neoplasm or death) and censored at the date of last follow-up for event-free patients. Overall survival (OS) was defined as the time from diagnosis to death from any cause or the date of last follow-up. Survival curves were estimated by the Kaplan-Meier method and compared by the log-rank test. Cumulative incidence of relapse (CIR) was calculated using cumulative incidence functions for competing events, constructed by the method of Kalbfleisch and Prentice and compared with Gray’s test. The Chi-squared test was used to compare proportions and the Kruskal-Wallis test to compare the equality of medians. All outcome rates are quoted at 5 years. In order to identify the HHD-profile with both the highest proportion and the best outcome, rankings for each HHD-profile were calculated. The sum of ranking performance of each profile for each endpoint (with 1 being the best and 8 the worst) was weighted by both the difference of survival compared to the ranked number one outcome and the proportion of the total and HHD B-ALL cohorts (Data Supplement):\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAA0AAAAAwCAYAAAA4qRoeAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAAFiUAABYlAUlSJPAAAByuSURBVHhe7Z3RaxzXFf+/+r03YbR5KqUUjfpgWhCkoxhaqeBCOqral5C2u3JCCMTYXT8UUrdeslKfGhvvtrEhFFkKNZjidFdNiktAWymBvMwSiqMYLaT0wZ6lhJCnWW/c/gH39/CbM7+7Z+/Mzq52pZX2fGCxNffOzJ1zzz333Dv3nplSSikIgiAIgiAIgiBMAP+HHxAEQRAEQRAEQTipyABIEARBEARBEISJQQZAgiAIgiAIgiBMDDIAEgRBEARBEARhYpABkCAIgiAIgiAIE4MMgARBEARBEARBmBhkACT0TbVaRS6Xw/T0NKampjA7O4tyucyzCYIgCIIgCMLYIQMgoW9WVlbg+z4ePHiAIAjwwx/+EIVCAZubmzyrIAiCIAiCIIwVMgASBmJrawuZTAaZTAZnz54FADx+/JhnEwRBEARBEISxQgZAE0Sj0cDU1NRAv3q9Hl1HKYWZmZno788//xwA8N3vfjc6dpyoVquYn5+XN1jHiFwuh1qtxg9HbG5uYn5+HtVqlScJQkSj0cD8/Dw/LAiCIJxwTsQAqFqtYnZ2NnLWTzrNZhMXL17E7OwsT+qA55ubm0OxWIzSNzY2oJQy/oIgQD6f167WTbPZRLlcxtraGiqVChYWFniWsabVamFpaQlra2u4dOkSLly4wLMIY0Yul0Oj0cDTTz+NJ554AuVyGaurqzwbLly4gEuXLmFtbQ1LS0totVo8izDB1Go1rK6uYm5uDplMJhoIcT1pNBrRHkeedlyo1+vI5XLI5XI8aSI4qZMhk16vwslmdnYWFy9eRLPZ5EkdtFotTE9PG/2AnqiUVCoVZVmWAmD8ua6rNjY2VBAE/NRDwff9qCwnGaqHfD6vfN/nyRFx+YIgiOrRsqye9WXbtgKgPM/rOF4qlTrqfnt7uyN93CE5OI7TUwZKqUhmSTIXRk8QBKpUKinXdZVt26pUKiXWXxAEynGcVLp+GGxsbCgAKp/P8yThkPE8TxWLRWVZlnJdt8vGEZ7nRfq2v7/Pk8caej7eToIgiNIAKMdxEp+N7GU2m+VJx4JKpaJs21au646FHTgoUq/CScf3fZXP55VlWapSqfDkDqhfjbPhcfQ1Wtje3jYOMra3t5XjOFGDOyoDYyrbSYIGHb2UoVe+SqUSyaqX4eulWL7vq2w2qwAcq0FQP06xLq+NjQ2eLBwivu8r13WV4zgqm83G6riOPtg9amhCwbIsniQcMhsbG9HAplgs9pzcyOfzibZw3HBdV1mWZXSAyUn2fV8FQRDljZNBNptNTD8OjNtkyKBIvQqTBPlfpVKJJ3Vg27aybbuvtt3XaMHzvNhBhv4G5qicxLiynQRo8NlLCdLmc103kldSh76/v98zTxAECuGboONA2gZFUCeCcIA/DtAgN6leTiKu66r9/X1VKpWU53lqY2MjVT32mhQ4DMh+ki6Nw4QBlSmNDE8SlUpFFYtFpUKdooF1r86zl0M5LhSLxVj7QH21XudJekBpR9WvD5NxmgwZBKlXYRKh/jupz+zXr1PDHAApzanu9VZhVCSV7bhj23aqmau0+fQBaz+jZt/3jQqGYzQAoln4NM9MA0D9LdA4OD/U1kwd4SSQzWYTjSGHBum2bfOkQyObzSrHcSInZhyWwVHHYmrTk8D+/n5fzjD1geNQd3GQbY+zx2TLuO2IO8e27b5kNO6Mw2TIIEi9CpMKTVz06r8ty1JWH6srRhIE4csvv+SHhANQrVbh+z7y+TwymQxPjkibDwBmZmZQKpUAAL7v49atWzyLkS+++AKFQiH68Gmr1Yr+/8tf/pLlHj8ajQZ830c2m+0pI4QbaG3bRi6XQzabBQB88MEHPNuh0mq1cO/ePX54oqhWq1heXuaHY8lkMshms/B9H41Ggyf3ZGpq6kAf+202m9ja2sKlS5fw85//HAif4aj58MMP+aGJYm5uDh9//DE/HMvCwgIcx8HNmzd7bs41US6XRx6o5w9/+AOQYI8/++wzfiiWcrkM3/dx48YNnnRseeWVVwAAa2trPGlgpF4FYXRkMhnk83n4vp/Yb+ZyObTb7cQ8OkMdAFGUnOeee44nodlsYnV1FfPz81G0ttnZ2a5QttVqFdPT0x0R3er1enTe9PR0X46Ifj+651HTbDY7ylWv19FqtXDx4sWOY8Tf//53AIgcpzjS5iNeeeUV2LYNACgUCqkcw1OnTqFYLOKdd97B1NQUnnrqKbzzzjvY3t7uyyEdFv3Kcnd3FwDw9NNPa1cx02q1UK1Wcf78eQDAmTNnAAB/+tOfWM5OKDoP6fH09DRyuVxHOShfXPRCXeeXlpY6ji8tLaHdbgMAFhcXO/Rbp1arYWlpqSN9aWmpK9w3DWJ1Oc7PzxsjX62urnZcC+EgkZ5jfn4+0iNeD7lcrut6BMmM8lIEmLj8g0B1TjrQD67r8kN98e6778KyLDz77LOYm5uDZVlot9td9k9Hj+Koy5wb92az2VHPup41m80OufLjJItCoRDl4degetTLMTs7i1wu1zUIqFarHWWJi+TTy87Pzs52PGe5XI7y6zrGIV3mMkuSc7+cO3cOGHAiZJBPBdRqtS470Wg0OuSsU61WYVnWge1xs9nEtWvXkM/nj12EzyQOOhliQupVEEYL+bXk55r4/ve/D/TI0wF/JZRE0hI4WipkWn5Fm0fz+XyURpvrYVhSRNdyXTeK3lIqlaLrIGYtYFzZ6Hjata76c/b746+fk6C9AL7vR0EkTNehY71Im09Hf1bTa/LjQlpZ9rN0jJYUkH7qywa5jhN0juM40Xme58Xu+yBdN9Ub7efS68X3feV5XvSMGxsbyvO86EfQMqtisaiCIFBBEETtR79eEG4MpmvRMcprWiJBbZfapOu6qlQqRdexwg26tNxLj0pkWjpEMjPZB9P9B4V0PY2ekx6RTN0w0iHJJs01dGzb7nh2ki/tQ+Hs7+9Hr/OpDLpum84jGXPdpuV/0HQsCALleV6Hbdb1iOrB9/1o6QFdV9dn/V5Jdt4y7JuJs/MUVIWu77quyufzqlQqRctXTUshSJfjZMbb3qD0o0cqbIvUDr2w7aqwfetyTYJk5YRRvUj+vF7TlI2WgPH78vNog3ycrTvODGPpp9SrIBwuvF1wdB8tDelyhVAj1C8eBIGqhCGX7ZgwoW64cZQ3OHJGTQMTug93mPROlsPLpjTnahzX+9Lzu2EIcWVYx5vG8Kk+8pnQHQ5TXRwH0shSz8c7CRNOGGmMH4vTJ3IWTbpOZTGlmfRW9ajTpOeIO48cYf04DZRMjgA5mzxNtwO6HAIWYl23BXERJHUHm0N6aZL1IMTJJQ4vHCAUi8VIF6wwZCx35pOgZzfJw/TcStMzXr+6gedpSTphkr1K4QjGXZNvxjYN1gmy2aY0Khe353RfGHQs7vmpTFxfyMmMk/UgxMnTRBAEiiLO0SDODftEXtYk6Bn0gTnJluhVn0prB7qtJ72iY1Sf9Pf+/n5UJ1b4eQVux44T/doCE1KvgnC4xPVHOghts2kswklnwUOogZl+vANLQ1KjpuvyxkiOpMlw0TkEzaj0Y4wOE6pM0/MTaQ112nwmyAkd9PxxII0sVcoGpFjwAx2a0eYDIz0tri2QjPlMNNdbIqlOk56jn4EDlcnk0Me9haFy8eOqRz2YnpPuYcpP9iFOnv2SJM8kqBxx8u5FNps1Ot8ke26oeznsVL/8LVCSTphkr3rY4F7l0EnSOboODPY87jiVy1RXcc9J8jRB9zHp+SDEyTMJ3/ejSYVsNtv1zL2ge/Ln1kmqTx2bhYwtlUrK0sInkzOvtP6B2iG1o2G1y6NgUFtgQupVEA6HONuvEzd5aGLgPUDh4Ame58GyLNy8ebNrbfow4BvVv/a1r3X8HUetVsOZM2ewvr4+9l9KHmT98DCZmZmJZPSDH/yAJx8rhiXLzc1NWJbVpTvPPvssAGBra6trf8rdu3cBAN/4xjc6jhPPPPMMAODTTz/lSUPl/fffB1K0lXq9Hu0lmpmZ4cn41re+BQDY29vjSYChbeqkrQeSGd+DMjU1hUKhAIRr1o8C2pNy//59OI6DYrGI119/HdVqtavu46DgB7/5zW94UqRbf/3rXzuO076cuP2KtJfpk08+4UlD5Z///CeQUA6dra0tIEbn5ubmov//+9//7kgj4nQprT3SdZnrkb6X4osvvtDOOhxoX5LjOMhms3AcB9PT0/jtb3870B6UYezb+Nvf/oZHjx7h9OnTmJ+fx7Vr17C+vo6ZmRncunULvu9HQXJu3bqFdruN3/3ud0B4/3w+P3AgiFERt3dmVEi9xkNBIfi+116Msg5Hee2jhJ5J3yuMA9QBtHMHPR+avJOo1+vRffrZ259EXF9iYuABELGwsID19XUAwMrKSmzDabVa2NzcRC6Xiza6koMzbKampvDjH/8Y7XYbjx8/5skCo9Fo4ObNm3AcB5cvX+bJHZxUI6JDwQ/y+TxPwszMDBzHAQbcBH0YkCN4nPA8L5pU4b+dnR1gSEY5Lc1mE6+++ipu3LiBarWKTCaD733ve3j77bfx2Wef4YUXXuCnGHn33XcBAD/96U95En7yk58A2uBh3DiOttN13S790X/kZMY5DaPg+vXrePz4MR48eIAf/ehHAID19XWcPXsW586dG7kum5ibm8OjR49w+/Zt3LhxA48ePYqCWhQKBRSLxWjg+sknn8C27Q7HgiZ5jmJAGcfbb78d2ebDYBLqVXdQeXvR7fGwHNhR1uEor32U+L7PDx2Yy5cvG/2fYbOwsIBKpcIPHxoHHgAhnMmkaGLFYpEno1qt4pvf/Cbu3r2Ll156CVtbW1BKRTMRw0Z/M/WLX/yi7zdTvNH38zsKo3cQWq0Wzp07B8uyUjlih2VEKErUqPjvf//LD0V88MEHaLfbUbhUDkWBSh1pROjJRx99xA91cVCjnFTnnJmZGXz88ccds7KffvopMpkMLl++HA3KevHWW2/FhqVfXl6GZVlDjUY16aSN8DcKpyGOK1eu4MqVK5EO0BvVhYWFLh07bBYWFjruf/HiRViWhV/96lfRsd3d3a63gF//+tcB1qao3zxIH3iQcNKZTMbYzkbFJNTrwsICPM8DANi2HU12I7TH5MPl8/mOydPLly93TDikZZR1OMprHyWm1Rs4QB0QcStZ0rKzswOlFD/chWnVwEFIuzoDwxoAAcDrr78OhLOZugFsNptYWVnB9PQ0dnZ2sLy8HFthw2RhYQHvvfceEDb+fhyMhYWFrpnDtL9Bla0XaSs1bT7i+vXr2Nvbi16T92LURqQehoZeWVkZyZsMWlKTtAztjTfegOu6sfKIWwb3ne98BwBw//796JjOw4cPgT6Wh33++ef8UCpogPqvf/2LJ3Vw6tSp6P+m9kH3P2gI6CRIZv/5z394kpGDGGWq87TLqnTSOtY6tVoNvu/j7NmzPCnCtAzu29/+NgDEfuuJZJX2OeLeyveiVzl0knROv/+o7KOuy2meN65tp6FfG6uTZqA/DAZ5e1er1bC7u4v19fWeNv6JJ57o+BdDerbD+i5VP5MhaRjGs6fhKOqV2uzs7GxXu6G+7CB2WRDSksb20kTEV77yFZ7UxdAGQLlcLnKUaDAE7VUqn2lotVp46623Oo4Nm4Xw9Vq73caZM2dSdYzjBhmfuH0YRNp8OrVaDVevXkU2m+3a63IU1Go1/OUvf8H7779vfJM4DEhH4zraRqOBvb09vPzyyzwpIm4ZHC2D4AMjhAM73/dh23asE6h/q6TZbKb6UJ/e8ZJ+/+xnPwMA/P73v+8qR6PRiPJlMplIHnwfCgDcvn0bABJlcVBIZjdv3jQOwg4ym8yhOh9kQKeU6rk8lPPnP/85sb4Rswzu9OnTsd8JouWZiFlWBwD/+Mc/ov+3Wq1UbUlvD61WC61Wq6Mc/NtRYPpKOmf6RhYtAzzI27te6Lpset5Wq2XUr0GgfUyD6BHNyo4Kckj73R/WarXw4osvwnXdrr7AcZxo8obgE0jlcjla0q5/m4zabyv8lpT+bTT+na+lpaVookFfVUHU6/WOa8zOzg5sHw4yGWLipNbrICQtVebf6EpaPUM6Q/oyjCV2RNK19WX+ern04+Vyues5y9q3ypaWloz2Rn9+fl9oer+6utrxnSg9X6PR6Pi2G/etEVMH+sqmcrnccf1Zwzc54zDdnz9H0laJWq3W8c3BfvwL/S2rCZK5ZVkde09j4VERktBD2Zqi6VAUEWhhFvWwraVSSXmepyqVinIcJ4pswqMZ6dfhEVUoChyPQKXfh5eNQko6jtN1vaOEysujgnEowlKvqBZp8ykWfriXTKjuCIrEwf+mX5r794J0Iy1pZam08M6m5yZdSfvj0eB0XSM99MLvplgsNDRB9WZZVvRtCdu2owhppihclIaw/dC3dijaD0VCsW1bFYtFVdK+r6LXpf7tCWqzgfYdIFNEIKob3gaVFoGFRysKtPDFce2TIhKVSqUo9DSYPtG96Rj9TT9+X4Lub5LlKNDtUdqfrht62HR6Vl/7po0p2pquEyRH27aja8Egez1CWzabjaJGUTvSz6V00k9LC+mu65weSlePJMjbW5Kdp5DWpihdcTLQdZn6F9J7y7K6rsWvT2WhXxykc3q44VGTJCsOyaAfSN5cP5TWPvX75vP5jvpXhrZJkG7oNpH0gtcJ71sIyq/7FXYYOlon7nwO5TPZ48PkONSrMrQVgsrP7a7JFyFbQnmpvfLrUt1kw88NBEEQXc/0HP2Q5tq6LdNtTBBG0dMj/el9ND0r9fdcN0m2lI/kodsRsseO40T3dsMw65RO/SSVIS4ysqkO6PqWZXW0JSf8fpouX1N75vcPNF9B1wGSFZi+mp7ZdL4J0jXuc+nQ9ZPy6KRuTZXwWz8IG6sd880fErpeiO3t7UihdOHpnatewSQ4ug9Vyvb2dkcZqMJN5+hlo3vT/dM4yaOGlympMyU58YEiJ20+1UcHQI6crpy8k6FGlc/nD2ygCGp8aehHlspgiHV0/Ur741TCAT6l2+FHMONkoxthaA4kNXiwARVBnRvCtqbXJRkm/Xmy2axR933f78rrhh+n5HA7oBtd/Rksy4rkS86KLg+ud2llZjLKVtiJ6sc4dJ7pmUaBPhhJ++P66Hlel0yz2Wzic9JAGGHdkJzpGrZtd+mAXqeO43Sl6/abrlEMPwCpEwSBKmkfK6XrlUqlrrwmm011XQoHYZSmd2Zke0gevD770WVKIzY2NiK95eXVIVlw3RwVJlkl6QA5FLwe4yD7zfWPIDuUDydDyAni+U1tU2ltgZeHdFu3BbxvITzP67qfKa/pGCc45MmQOI5LvSrD5AD/8XNMumCqm6RjvZxxKn+vn6kMva5Nk/263SBfSD+XysDrhOqKdJvqgtshWwtLTsAwmCGozXD7ZDrH9FwqzMt9RPKJdN/JdH7c/a1wglfHVLeWYWBIMuQ6xCF/h8tQh8qXlEcn2VIIY4PNZlzjSJOPFLuXwqmYzktXbHJceSNTKYxmUhmojKPCNOMhjD+6UfY8T7nah2/j8MO3ndzwCpMNQqeBBm6u6/a0B9xpHEfIUeMOURyu6yq7x0qA7e3tyFmPc5JNDpOKmYlWMflNThPheZ4qFotRHvrpJJ1P0H3TOknjwlHVq4pxsFWC82qqW9IDHVN9mY6ZrjcI/VzbZm8YS6VSV7un5+fn8mv2mhDTQYysVcwAQsWcw8tAwFBfpno0nU8TSxyTXPkxGgTywZfp3pwg/Mh60qTFIBMbQ9sDJIyWN998E+12G9evX+dJHfTK12g0UCgU4Lpuzz0N9Xodr732GsA2RRJTU1NYWVnB/Py8cU1mOMBO/PUqw6igPRfZbLZrn4ww/iwuLmJxcRGtVit2LwzCtd7ZbBYY43DTwtGxu7uLp556CoVCAS+//HLXJm/Oq6++Csuy8Otf/5onjQ0zMzMoFovY3d1Nta5/Z2cHDx8+TNwgv7y8jIcPH0IphUePHvVlt7/88kt+qG+q1SoWFxfx5JNPolQqQSk10B6sZrOJa9euwXGcrj0x48641Wu/vPTSS0C4PwWhL3Lv3r2R7g08COfPn8fe3h4ajQZarRauXbuWGNAmCQpeEfe5h7S02+3E+hw1BwlM9b///Q8A8OSTT/Kknly/fh3tdhtvvvkmT4qg/djnz5/nSbHIAOiYsLy8jGKxiKtXryaG9U7K12q18PzzzwNhx0+b0OJ+i4uLiQqvlIo+nLa6usqTx5qZmRk8ePAACDedc1kJ443neahUKtjb24v9lku1WsXp06cBAA8ePOjp3AqTh+u6CIIAjuNgZWUl0bG8ePEi9vb2cOfOnbHXpStXrsBxHLz44ovGzdiHCQUa4FEtKZrhV7/61Y7jJm7fvh19py7V5mYDJ2EyZJzqtV9Onz4Nx3Hw4YcfYmpqCmfOnEE+n48+xDpu0GcwNjc3cevWLTzzzDPGiV4Yogpy3aYgFqYomf1MwLquO1BEUg6PJkhtk8Kgx0GTDjyg2MOHD6NP4cRBsosLQBVHtVrF1atXUSwWsby8zJMj1tbWYNt27OdLjPBXQsJ4Q2s1TfsjdEz59D0j/f7ilikEMRsG1f+b1uj5i3vtSa9fDwNaxsfLL4wf/LU8rbXmSxOkToVegO0jpXXsfH+aFy61NKWNO7QfqtRjX9MwoH0TZNM9z1O+70dLUG1t7x/l5e2W+ihq39R+aTkX9WUbGxtde7H0vojXU6VSiZY0jVoOh8Fh1istUTLJjpZ28Xoku6z7DdlstmuZFieuDul6B7Hng1yb5GxpwQt0SDZuuJRWP6bvXVTasnu6jh/uV9R9IFrmaJK1Yn6dCp+J9n3q7UvF1IEK7Z6lLf/3wz26fLkkna8vMad2qz+vaW+OLmsdLmvf96OlkdkwMAVB8uHXNkFy4XuxenE4HqYwVEgxTGtBddLm6wddsfUGRGtDe+3HSIPv+9Egq1+FFk42JoNI+pjXIuMIQhImR4M6d+6k2LZ9KI7mqPDCYBrcSR0F1D6tMKIlyWx/f78joAfJlKPbfjuMhEnn014VOwzkQZMhCJ0sOk8/pk7wZMhh1Cs58/TTBzC6/KENfPXjrravjpxU/nMcJ3LcTXXIy8Ad+rQMcm19gGOC0qkPgkH3iYAFJtL1m9DLEnfPkhYkxg0D3dAEAZ0TVwcqvIcd7hWnPHzwwetWlwsPiuMYAufostb1k2SgpwXh3h3HcTr2BzmOo/I9JvqVtj9okHYgAyChL0xGRLG3S6aOLS36tfXfQa4pnAzijDI5rkjoNARBR9cjXWf0jp07Q4IgDA45tDqBFlp6HCHnPM4W0AAoLn0cEX/q/zOl+tmBJQiCIAiCIAgpqdfrWFxchOd5XftoaA/nzs5Ox/FxYHNzE3fv3o0tW9JzjStTU1MolUojDXhxXJAgCIIgCIIgCMJIOHXqFCzLwh//+MeODfTVahX37t3Dc88915H/KCmXyyiXy2g0GnjttddQKpV4loiPPvqo499xp16vAwDu37/fV/CFk4oMgARBEARBEISRkMlk8N577wEAbNuOIs2+8cYbuHPnDi5cuMBPOVIKhQKef/553LlzJzbqYL1eR6FQAML8FN57nFlcXATCKIgvvPACT544ZAmcIAiCIAiCIAgTg7wBEgRBEARBEARhYpABkCAIgiAIgiAIE4MMgARBEARBEARBmBhkACQIgiAIgiAIwsQgAyBBEARBEARBECYGGQAJgiAIgiAIgjAxyABIEARBEARBEISJQQZAgiAIgiAIgiBMDDIAEgRBEARBEARhYpABkCAIgiAIgiAIE8P/BbidgP7MQI56AAAAAElFTkSuQmCC\" width=\"609\" height=\"48\"\u003e\u003c/p\u003e\n\u003cp\u003e(*: best performing; Δ: difference; r: ranking; k1-3: 5-year EFS, OS, and CIR).\u003c/p\u003e\n\u003cp\u003eEach individual study group performed all statistical analyses according to the pre-defined definitions ensuring consistency. While we did not centralize individual patient data for this study, summary statistics were collected for all eight trials using a standardized form (Data Supplement). P-values ≤0.05 were considered statistically relevant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eOverall, 11,530 B-ALL patients treated across eight trials from seven study groups were included in this analysis (Table 1). Sex ratio, median age, median leukocyte counts, and National Cancer Institute (NCI) risk distribution for the HHD (n = 3,231) and non-HHD B-ALL (n = 8,299) patients enrolled into the respective trials are shown in Table 1. The frequency of HHD among B-ALL cases varied significantly from 19% in ALL-IC BFM 2009 to 35% in EORTC-CLG 58951 (p \u0026lt; 0.001; Fig. 1A) [22, 34]. However, there was no statistically significant difference with respect to sex, age, leukocyte count or NCI risk among the HHD B-ALL cases across the eight trials (Fig. 2). Hence, the difference in frequency more likely reflects differential cytogenetic success rates rather than genuine biological differences.\u003c/p\u003e\n\u003cp\u003eInitially, we compared the outcome of B-ALL patients with and without HHD per trial. In six of eight trials, HHD patients had a lower 5-year CIR compared to other B-ALL patients, but the difference was significant in only three trials (Fig. 1B). In the remaining two trials – ALL-BFM-Austria (A) 2000 and Dutch Childhood Oncology Group (DCOG) ALL10 – HHD patients had a non-significant higher CIR compared to other B-ALL patients [32, 33]. The same pattern was observed for 5-year EFS and OS rates, where four trials reported significantly superior rates, two trials non-significant higher rates and two trials (ALL BFM-A 2000, DCOG ALL10) slightly lower rates for HHD patients (Fig. 1C/1D) [32, 33].\u003c/p\u003e\n\u003cp\u003eSeveral studies have proposed subgroups of HHD ALL, based on the pattern of specific chromosome gains or MCN, which have superior outcomes [9, 11, 14–16, 26, 40, 41]. Hence, we classified all 3,231 patients with HHD B-ALL into eight subgroups using consistent definitions based on cytogenetic karyotypes. The frequency of DT, TT, T18 and UK-GR profile varied between each trial (Fig. 3A). However, the pattern of variation was consistent for each subgroup (\u003cem\u003ei.e.\u003c/em\u003e, the same trial had the lowest frequency of each subgroup), suggesting that the differences were driven by the quality of the cytogenetic data (\u003cem\u003ei.e.\u003c/em\u003e, number of marker chromosomes) rather than by genuine biological heterogeneity. Consistent with this explanation, the frequency of the MCN subgroups (51–53, 54–55, and 56–67) was much more comparable across the trial datasets (Fig. 3B).\u003c/p\u003e\n\u003cp\u003eTo assess the potential clinical utility of each subgroup of HHD, we considered two key metrics: (1) the 5-year outcome measures, and (2) the proportion of B-ALL patients captured (Table 1). We opted for the proportion of B-ALL rather than the proportion of HHD B-ALL due to the variability in HHD-frequency across the trial datasets (Fig. 1A). To visualize the two metrics for all eight definitions across all eight trials, we plotted the 5-year outcome measures against the B-ALL proportion for 5-year CIR, EFS and OS (Fig. 4). In each plot, there is evidence of clustering, with the UK-GR profile, T18 and MCN 54–67 subgroups tending to have the highest frequency and best outcome. Intriguingly, the plots also showed the subgroup of MCN 51–53 to have the lowest frequency and worst outcome. To compare each subgroup within each trial, we developed a ranking system, which considers the difference in outcome for each endpoint as well as the size of the putative subgroup using both the total B-ALL and HHD B-ALL cohorts as reference points (Fig. 5A). Using this system, all eight subgroups were ranked from one to eight for each trial separately (Fig. 5B). As a next step, we summed the ranks across the eight trials so that each subgroup was assigned an overall score of between eight (always ranked first) to 64 (always ranked last). As a result, the two highest-ranking subgroups were MCN 54–67 and UK-GR profile with overall scores of 16 and 17, respectively (Fig. 5C). Patients with MCN 51–53 had the worst outcome in all trials and, hence, achieved an overall score of 64. The remaining subgroups had overall scores of between 28 and 51. Accordingly, apart from MCN 54–67 (ranked #1 in 5 trials) and UK-GR profile (ranked #1 in 2 trials), only T18 reached rank #1 (overall score: 28) in one trial (UKALL2003). Moreover, the second worst subgroup was MCN 54–55 (ranked #7 in 5 trials) with an overall score of 51.\u003c/p\u003e\n\u003cp\u003eTo further explore the UK-GR profile and MCN 54–67, we evaluated outcomes among patients enrolled on the eight trials who were classified in either one, both, or neither subgroup. Among 712 UKALL2003 HHD B-ALL patients, the majority (n = 493; 69%) were positive for both UK-GR and MCN 54–67, with a 5-year EFS of 93.9% (95% CI, 91.3–95.7). Patients with UK-GR profile only (n = 75) had a 5-year EFS of 90.7% (95% CI, 81.4–95.4), whereas those with MCN 54–67 profile only (n = 93) had a 5-year EFS of 83.6% (95% CI, 74.3–89.8). Patients belonging to neither subgroup (n = 51) had a 5-year EFS of 86.2% (95% CI, 73.2–93.2). An analogous analysis was performed for the 843 HHD B-ALL patients from trial AIEOP-BFM ALL 2009. The corresponding 5-year EFS rates were 91.3% (95% CI, 88.7–93.3), 89.1% (95% CI, 80.7–94.0), 91.9% (95% CI, 84.5–95.9), and 73.1% (95% CI, 57.5–83.7) for patients positive for both UK-GR and MCN 54–67 (n = 603, 72%), UK-GR only (n = 93), MCN 54–67 only (n = 102), and neither subgroup (n = 45), respectively. Analyses of the other six trials, each comprising fewer HHD patients than UKALL2003 and AIEOP-BFM ALL 2009, revealed that – with the exception of ALL-IC BFM 2009 – the vast majority of HHD cases were positive for both the UK-GR and MCN 54–67 profiles. The latter consistently demonstrated the most favorable EFS rates. In contrast, outcomes for the remaining three subgroups showed no consistent pattern across the six trials (Data Supplement).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present i-BFM ALL study reports on a large cohort of 3,231 children and adolescents with HHD ALL out of 11,530 patients with B-ALL. These HHD cases were consistently classified into eight potential low-risk subgroups, defined by either specific chromosomal gains or distinct categories of MCN [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Within this multinational study framework, we clearly demonstrated that both an MCN of 54\u0026ndash;67 and the UK-GR profile represent robust genetic markers identifying the largest HHD subgroups (up to 28% for MCN 54\u0026ndash;67 and 26% for UK-GR) with the most favorable outcomes among total B-ALL cases. Five-year EFS ranged from 80.5\u0026ndash;92.8% for MCN 54\u0026ndash;67 and from 80.5\u0026ndash;93.5% for UK-GR profile across our trial datasets. Importantly, both subsets are easy to detect in clinical trial settings \u0026ndash; regardless of whether conventional G-banded karyotyping or more advanced methodologies are employed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. While the MCN requires only assessment of the total chromosome count, the UK-GR profile is based on assessment of four specific chromosomes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These findings could be particularly relevant for resource-limited settings, where conventional cytogenetics (or DNA index), sometimes complemented by fluorescence \u003cem\u003ein-situ\u003c/em\u003e hybridization, remains the standard diagnostic approach for HHD [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe data collected from the individual trials revealed a substantial variation in the frequency of HHD among their total B-ALL cohorts, ranging from 19% to 35%. As there were no relevant differences with regard to basic demographics such as sex, age or leukocyte counts at diagnosis, this variability confirms that conventional G-banded karyotyping has inherent limitations overlooking HHD cases [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, implementation of newer technologies, such as SNP arrays or next-generation-sequencing-based approaches may improve the accurate detection of HHD and its clinically relevant subsets in the future [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWith the emergence of less toxic immunotherapies such as blinatumomab, next-generation pediatric low (standard)-risk ALL trials aim to achieve substantial treatment de-escalation while maintaining high cure rates of 90\u0026ndash;95% in this subset [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous studies have primarily focused on identifying intermediate- and high-risk genetic abnormalities (\u003cem\u003ei.e.\u003c/em\u003e, \u003cem\u003eIKZF1\u003c/em\u003e\u003csup\u003eplus\u003c/sup\u003e, \u003cem\u003eIKZF1\u003c/em\u003e-deletion, hypodiploidy, iAMP21, \u003cem\u003eTCF3\u003c/em\u003e- or \u003cem\u003eKMT2A\u003c/em\u003e-rearrangements) to guide therapy intensification and improve outcomes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45 CR46 CR47 CR48\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In contrast, good-risk genetics such as \u003cem\u003eETV6::RUNX1\u003c/em\u003e or HHD have, at least in some European-based protocols, been neglected in recent decades for risk stratification and been utilized to support treatment de-intensification in only a few studies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Interestingly, HHD was used to define the very low-risk group in our included EORTC-CLG 58951 trial and, yet, retained a significant favorable prognostic impact (MCN 54\u0026ndash;67 ranked #1) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo address the objectives of the present study \u0026ndash; namely, to identify the HHD subgroup with both the highest proportion of B-ALL patients and the most favorable outcome \u0026ndash; we implemented a standardized approach across participating study groups. Each group was asked to provide not only general cohort characteristics, but also fully analyzed outcome data stratified according to the predefined eight HHD B-ALL subgroups, as compared with their B-ALL counterparts. This approach avoided collection of individual patient-level data from \u0026gt;\u0026thinsp;11,500 patients, while allowing simultaneous evaluation of CIR, EFS and OS for all eight subgroups and their relative frequencies among total B-ALL cases. Ranking across the eight trials identified MCN of 54\u0026ndash;67 and UK-GR profile as the largest subgroups with the best prognoses, as reflected by the lowest cumulative scores (16 and 17, respectively). Conversely, the MCN 51\u0026ndash;53 subgroup exhibited the highest score possible (64), thus, representing the smallest subgroup (3\u0026ndash;7%) with the poorest outcomes (EFS ranged from 68.7\u0026ndash;88.9%).\u003c/p\u003e\u003cp\u003eA recent study by the St. Jude Children\u0026rsquo;s Research Hospital (SJCRH) conducted a comparable analysis of 915 B-ALL patients enrolled in two consecutive frontline trials, using six different HHD-definitions: (1) MCN 51\u0026ndash;67, (2) DNA index 1.16\u0026ndash;1.6 (with \u0026ge;\u0026thinsp;1.16 corresponding to MCN\u0026thinsp;\u0026ge;\u0026thinsp;54 and 1.6 to approximately 74 chromosomes), (3) UK-GR profile, (4) T18, (5) DT, and (6) TT [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In multivariable analysis, only the DNA index of 1.16\u0026ndash;1.6 \u0026ndash; comprising 25.3% of all patients \u0026ndash; remained as an independent prognostic marker for CIR and EFS [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These findings suggested that the DNA index of 1.16\u0026ndash;1.6 might represent the most clinically practical HHD-definition in the context of SJCRH-based therapies, as it identifies a sufficiently large subgroup with excellent outcomes and combines favorable prognostic power with low cost, technical simplicity, and rapid turnaround. The main difference between the SJCRH and our analysis lies in methodology: we did not analyze HHD according to DNA index, whereas the SJCRH study did not perform separate analyses according to our MCN subgroups [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Specifically, the SJCRH study\u0026rsquo;s inclusion of patients with 51\u0026ndash;53 chromosomes within the broader MCN 51\u0026ndash;67 group likely diluted outcome measures, as these lower-MCN cases are associated with inferior prognosis (at least in our dataset). On the top of their prognostic factor analysis, the SJCRH study nicely demonstrated that \u003cem\u003eex-vivo\u003c/em\u003e drug sensitivities are associated with gain of specific chromosomes (\u003cem\u003ei.e.\u003c/em\u003e, trisomy 17 was significantly associated with L-asparaginase and 6-mercaptopurine sensitivity) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEvident limitations of our multinational study include the retrospective nature of data collection over a 20-year period also encompassing trials initiated before 2000 (ALL-BFM-A 2000, EORTC-CLG 58951) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Some of our participating groups may have missed cases of HHD B-ALL due to failed karyotyping or suboptimal cytogenetic quality. Moreover, chemotherapy protocols and risk stratification criteria varied among the participating trials.\u003c/p\u003e\u003cp\u003eIn conclusion, an MCN of 54\u0026ndash;67 and the UK-GR profile define large subgroups of HHD B-ALL with very favorable prognosis. Both can serve as practical genetic markers to prospectively stratify children with B-ALL into low-risk trials, where substitution of conventional chemotherapy with immunotherapy and systematic reduction or modification of chemotherapeutic components can be evaluated in randomized settings [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Beyond genetic classification, early treatment response as assessed by minimal residual disease (MRD) remains the most critical predictor of relapse in childhood ALL [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Accordingly, the interplay between HHD and treatment response is planned for assessment in the upcoming AIEOP-BFM ALL 2025 trial for standard-risk B-ALLs defined by MCN 54\u0026ndash;67 and MRD negativity at end-of-induction [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast, patients with an MCN of 51\u0026ndash;53 appear to form a numerically related, yet biologically distinct form of HHD, and, thus, not to be ideal candidates for treatment de-intensification but requiring further molecular characterization to elucidate the biological mechanisms underlying their inferior outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA SUPPLEMENT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors retain all rights in any data supplements associated with their articles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon request to AA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAA and KN were supported by St. Anna Children’s Cancer Research Institute; SS, GC, AM, MS, and MZ were supported by Deutsche Krebshilfe e.V. (funding number 108588) for the AIEOP-BFM ALL 2009 trial; MC was supported by Chilean National Pediatric Cancer Program; RP and MLB received core funding from KiKa and JMB was supported by KiKa grant 264; LC's fellowship at the EORTC Headquarters was supported by the EORTC Cancer Research Fund (ECRF); CK and EZ were supported by NKFIH-OTKA K 143021 [Foundation for Children with Leukemia (Leukemias Gyermekekert Alapitvany)].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAA: Honoraria: Jazz Pharmaceuticals, Amgen, Novartis; Consulting or Advisory Role: Jazz Pharmaceuticals, Amgen, Novartis, Takeda Science Foundation; Travel, Accommodations, Expenses: Jazz Pharmaceuticals\u003c/p\u003e\n\u003cp\u003eSS: Consulting and Advisory Role: SERB Pharmaceuticals, Jazz Pharmaceuticals\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGC: Research support: Amgen, Clinigen, Jazz Pharmaceuticals, Servier; Travel, Honoraria: Amgen, Clinigen, Jazz Pharmaceuticals\u003c/p\u003e\n\u003cp\u003eSE: Consulting or Advisory Role: Jazz Pharmaceuticals\u003c/p\u003e\n\u003cp\u003eAM: Travel: Jazz Pharmaceuticals; Consulting or Advisory Role: Jazz Pharmaceuticals\u003c/p\u003e\n\u003cp\u003eMS: Research support: Shire, Clinigen, Jazz Pharmaceuticals, Servier, Amgen; Honoraria: Servier, Amgen, Clinigen, Jazz Pharmaceuticals\u003c/p\u003e\n\u003cp\u003eAll other authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: AA, AE, AVM and MZ. Provision of study materials or patients: AA, CA, DB, LB, NB, JMB, MC, GC, HC, LC, SE, GE, HAG-K, MLB, CK, JL, AM, HM, KN, RP, PR, MS, AV, EZ, JZ, AB, MZ and AVM. Collection and assembly of data: AA, CA, DB, LB, NB, JMB, MC, GC, HC, LC, SE, GE, HAG-K, MLB, CK, JL, AM, HM, KN, RP, PR, MS, AV, EZ, JZ, AB, MZ and AVM. Data analysis and interpretation: AA, AE, MZ, HAG-K, LC, and AVM.\u0026nbsp;Manuscript writing: All authors. Final approval of manuscript: All authors. Accountable for all aspects of the work: All authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all participating institutions, geneticists, biologists, and physicians for their support of the study. This i-BFM ALL Study Group paper was written on behalf of the Berlin-Frankfurt-Münster (BFM) Study Group (Austria, Germany, Switzerland, Czech Republic, Switzerland, Israel, and Australia/New Zealand), Cooperative Study Group for ALL (COALL; Germany), Dutch Childhood Oncology Group (DCOG; The Netherlands), European Organization for Research and Treatment of Cancer – Children’s Leukemia Group (EORTC-CLG; France, Belgium, and Portugal), United Kingdom Children’s Cancer and Leukemia Study Group (CCLG) and ALL-IC (Intercontinental) BFM Study Group (Argentina, Chile, Croatia, Cuba, Hungary, Serbia, Slovakia, Slovenia, Poland, Moscow, Turkey, Ukraine (Kiev) and Uruguay).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHunger SP, Mullighan CG. Acute Lymphoblastic Leukemia in Children. N Engl J Med. 2015;373:1541-1552.\u003c/li\u003e\n\u003cli\u003ePui CH, Yang JJ, Hunger SP, Pieters R, Schrappe M, Biondi A, et al. 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A report from the LALA-94 and LALA-SA groups on hypodiploidy with 30 to 39 chromosomes and near-triploidy: 2 possible expressions of a sole entity conferring poor prognosis in adult acute lymphoblastic leukemia (ALL). Blood. 2004;104:2444-2451.\u003c/li\u003e\n\u003cli\u003eJackson JF, Boyett J, Pullen J, Brock B, Patterson R, Land V, et al. Favorable prognosis associated with hyperdiploidy in children with acute lymphocytic leukemia correlates with extra chromosome 6. A Pediatric Oncology Group study. Cancer. 1990;66:1183-1189.\u003c/li\u003e\n\u003cli\u003ePurvis K, Zhou Y, Karol SE, Rubnitz JE, Ribeiro RC, Lee S, et al. Outcomes in patients with ETV6::RUNX1 or high-hyperdiploid B-ALL treated in the St. Jude Total Therapy XV/XVI studies. Blood. 2025;145:190-201.\u003c/li\u003e\n\u003cli\u003eAttarbaschi A, Mann G, Konig M, Dworzak MN, Trebo MM, Muhlegger N, et al. 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Nat Genet. 2022;54:1376-1389.\u003c/li\u003e\n\u003cli\u003eChang TC, Chen W, Qu C, Cheng Z, Hedges D, Elsayed A, et al. Genomic Determinants of Outcome in Acute Lymphoblastic Leukemia. J Clin Oncol. 2024;42:3491-3503.\u003c/li\u003e\n\u003cli\u003ePui CH, Rebora P, Schrappe M, Attarbaschi A, Baruchel A, Basso G, et al. Outcome of Children With Hypodiploid Acute Lymphoblastic Leukemia: A Retrospective Multinational Study. J Clin Oncol. 2019;37:770-779.\u003c/li\u003e\n\u003cli\u003eStanulla M, Dagdan E, Zaliova M, Moricke A, Palmi C, Cazzaniga G, et al. IKZF1(plus) Defines a New Minimal Residual Disease-Dependent Very-Poor Prognostic Profile in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia. J Clin Oncol. 2018;36:1240-1249.\u003c/li\u003e\n\u003cli\u003eMoorman AV, Richards SM, Robinson HM, Strefford JC, Gibson BE, Kinsey SE, et al. Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21). Blood. 2007;109:2327-2330.\u003c/li\u003e\n\u003cli\u003eDomenech C, Kicinski M, De Moerloose B, Piette C, Chahla WA, Kornreich L, et al. Results of the prospective EORTC Children Leukemia Group study 58081 in precursor B- and T-cell acute lymphoblastic leukemia. Hemasphere. 2024;8:e70025.\u003c/li\u003e\n\u003cli\u003eGottschalk H, Moricke A, Conter V, Schrappe M, Stary J, Cario G, et al. Reducing Daunorubicin in Induction Therapy in Children With B-Lineage ALL With Favorable Prognosis: Results of Phase III Trial AIEOP-BFM ALL 2009. J Clin Oncol. 2025;JCO2501357.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Connor D, Enshaei A, Bartram J, Hancock J, Harrison CJ, Hough R, et al. Genotype-Specific Minimal Residual Disease Interpretation Improves Stratification in Pediatric Acute Lymphoblastic Leukemia. J Clin Oncol. 2018;36:34-43.\u003c/li\u003e\n\u003c/ol\u003e "},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8252282/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8252282/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eB-cell acute lymphoblastic leukemia (B-ALL) with high-hyperdiploidy (HHD) represents the most prevalent genetic subtype of childhood ALL. This retrospective international study aimed to identify the HHD subgroup(s) having both the highest proportion of B-ALL cases and the best outcome.\u003c/p\u003e\u003cp\u003eData from 11,530 B-ALL patients, including 3,231 with HHD-B-ALL, treated between 1998 and 2018 in eight trials from seven international Berlin-Frankfurt-M\u0026uuml;nster Group members were analyzed. Eight HHD subgroups \u0026ndash; including double and triple trisomies, trisomy 18, UK-good-risk (UK-GR) profile, and four different modal chromosome number (MCN) categories \u0026ndash; were evaluated for 5-year event-free survival (EFS), overall survival, and relapse. Each subgroup was ranked by outcome measures and prevalence within the B-ALL trial cohorts.\u003c/p\u003e\u003cp\u003eHHD frequency ranged from 19\u0026ndash;35%. MCN 54\u0026ndash;67 and UK-GR profile achieved the lowest overall ranking scores corresponding to the largest (up to 28% for MCN 54\u0026ndash;67 and 26% for UK-GR among total B-ALL trial cohorts) and best HHD subgroups (EFS ranged from 80.5\u0026ndash;92.8% for MCN 54\u0026ndash;67 and 80.5\u0026ndash;93.5% for UK-GR).\u003c/p\u003e\u003cp\u003eBoth an MCN of 54\u0026ndash;67 and UK-GR profile define large, reproducible subgroups of HHD B-ALL with good prognosis. They represent strong candidates for risk-stratification of treatment de-intensification trials in the modern immunotherapy era.\u003c/p\u003e","manuscriptTitle":"Identification of favorable subgroups of childhood high-hyperdiploid acute lymphoblastic leukemia in a modern therapy era – A retrospective international study of 3,231 patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 11:02:59","doi":"10.21203/rs.3.rs-8252282/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cb0f476f-82ef-427e-bca7-175ed94d21c0","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59412053,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Leukaemia/Acute lymphocytic leukaemia"},{"id":59412054,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2026-01-14T15:12:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 11:02:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8252282","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8252282","identity":"rs-8252282","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}