ORIGINAL RESEARCH published: 05 February 2021 doi: 10.3389/fped.2020.614521 Measurable Residual Disease Edited by: Assessed by Flow-Cytometry Is a Yong-mi Kim, Children’s Hospital of Los Angeles, United States Stable Prognostic Factor for Reviewed by: Pediatric T-Cell Acute Lymphoblastic Elizabeth Macintyre, Université de Paris, France Arnaud Petit, Leukemia in Consecutive SEHOP Assistance Publique Hopitaux De Paris, France Protocols Whereas the Impact of Maddalena Paganin, University of Padua, Italy Oncogenetics Depends on Treatment *Correspondence: Mireia Camós Nerea Vega-García 1,2*, Sara Perez-Jaume 3 , Elena Esperanza-Cebollada 1,2 , mcamos@sjdhospitalbarcelona.org Clara Vicente-Garcés 1,2 , Montserrat Torrebadell 1,2,4 , Antonio Jiménez-Velasco 5 , Nerea Vega-García Margarita Ortega 6 , Marta Llop 7,8 , Lorea Abad 9 , José Manuel Vagace 10 , nvega@sjdhospitalbarcelona.org Alfredo Minguela 11 , Marta Pratcorona 12 , Joaquín Sánchez-Garcia 13 , Clara B. García-Calderón 14 , María Teresa Gómez-Casares 15 , Estela Martín-Clavero 16 , Specialty section: Adela Escudero 17 , Marta Riñón Martinez-Gallo 18 , Luz Muñoz 19 , María Rosario Velasco 20 , This article was submitted to Marina García-Morin 21 , Albert Català 2,4,22 , Antonia Pascual 23 , Pablo Velasco 24 , Pediatric Hematology and Hematological Malignancies, José Mª. Fernández 25 , Alvaro Lassaletta 26 , José Luis Fuster 27 , Isabel Badell 28 , a section of the journal Águeda Molinos-Quintana 14 , Antonio Molinés 29 , Pilar Guerra-García 30,31 , Frontiers in Pediatrics Antonio Pérez-Martínez 17,31 , Miriam García-Abós 32 , Reyes Robles Ortiz 33 , Sandra Pisa 34 , Rosa Adán 35 , Cristina Díaz de Heredia 24 , José Luis Dapena 2,22 , Susana Rives 2,4,22 , Received: 06 October 2020 Manuel Ramírez-Orellana 9 and Mireia Camós 1,2,4* on behalf of the Biological Accepted: 31 December 2020 Published: 05 February 2021 Committee of the Group of Leukaemia of the Spanish Society of Paediatric Haematology Oncology (SEHOP Group) Citation: Vega-García N, Perez-Jaume S, 1 Haematology Laboratory, Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain, 2 Developmental Tumor Esperanza-Cebollada E, Biology Group, Leukemia and Other Pediatric Hemopathies, Institut de Recerca Sant Joan de Déu, Barcelona, Spain, Vicente-Garcés C, Torrebadell M, 3 Developmental Tumour Biology Laboratory, Institut de Recerca Hospital Sant Joan de Déu Barcelona, Barcelona, Spain, Jiménez-Velasco A, Ortega M, Llop M, 4 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain, Abad L, Vagace JM, Minguela A, 5 Haematology and Hemotherapy Laboratory, Hospital Carlos Haya, Málaga, Spain, 6 Cytogenetics Unit, Hematology Pratcorona M, Sánchez-Garcia J, Department, Hospital Vall d’Hebron, Barcelona, Spain, 7 Molecular Biology Unit, Clinical Analysis Service, La Fe University García-Calderón CB, and Polytechnic Hospital, Valencia, Spain, 8 Centro de Investigación Biomédica en Red - Cáncer (CIBERONC Gómez-Casares MT, Martín-Clavero E, CB16/12/00284), Madrid, Spain, 9 Paediatric Hemato-Oncology Laboratory, Hospital Niño Jesús, Madrid, Spain, Escudero A, Riñón Martinez-Gallo M, 10 Haematology Laboratory, Hospital Materno Infantil, Badajoz, Spain, 11 Immunology Service, Clinic University Hospital Virgen Muñoz L, Velasco MR, de la Arrixaca (HCUVA) and Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain, 12 Haematology Laboratory, García-Morin M, Català A, Pascual A, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 13 Hematology Department, Hospital Reina Sofía, IMIBIC, UCO, Velasco P, Fernández JMa , Córdoba, Spain, 14 Instituto de Biomedicina de Sevilla (IBIS/Consejo Superior de Investigaciones Científicas (CSIC)/Centro de Lassaletta A, Fuster JL, Badell I, Investigación Biomédica en Red - Cáncer (CIBERONC)), Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Molinos-Quintana Á, Molinés A, Seville, Spain, 15 Biology and Molecular Haematology and Hemotherapy Service, Hospital Universitario de Gran Canaria Guerra-García P, Pérez-Martínez A, Doctor Negrín, Las Palmas de Gran Canarias, Spain, 16 Haematology-Cytology Department, Hospital Universitario 12 de García-Abós M, Robles Ortiz R, Octubre, Madrid, Spain, 17 Translational Research in Pediatric Oncology Hematopoietic Transplantation and Cell Therapy, Pisa S, Adán R, Díaz de Heredia C, Institute of Medical and Molecular Genetics (INGEMM), Hospital La Paz Institute for Health Research (IdiPAZ), Madrid, Spain, Dapena JL, Rives S, 18 Immunology Laboratory, Hospital de Cruces, Bilbao, Spain, 19 Haematology Laboratory, Hospital Parc Taulí, Sabadell, Ramírez-Orellana M and Camós M Spain, 20 Haematology Department, Hospital Virgen de la Salud, Toledo, Spain, 21 Paediatric Hematology Unit, Hospital (2021) Measurable Residual Disease General Universitario Gregorio Marañón, Madrid, Spain, 22 Paediatric Hematology and Oncology Departments, Hospital Sant Assessed by Flow-Cytometry Is a Joan de Déu, University of Barcelona, Barcelona, Spain, 23 Haematology Department, Hospital Carlos Haya, Málaga, Spain, Stable Prognostic Factor for Pediatric 24 Pediatric Hematology and Oncology Department, Hospital Universitari Vall d’Hebron, Barcelona, Spain, 25 Haematology T-Cell Acute Lymphoblastic Leukemia and Oncology Department, Hospital de La Fe, Valencia, Spain, 26 Haematology and Oncology Department, Hospital Niño in Consecutive SEHOP Protocols Jesús, Madrid, Spain, 27 Paediatric Oncohematology Department, Clinic University Hospital Virgen de la Arrixaca (HCUVA) Whereas the Impact of Oncogenetics and Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain, 28 Paediatric Hematology Department, Hospital de Depends on Treatment. la Santa Creu i Sant Pau, Barcelona, Spain, 29 Unit of Hematology and Hemotherapy, H.U. Materno Infantil de Canarias, Front. Pediatr. 8:614521. Canarias, Spain, 30 Paediatric Hemato-Oncology Department, Hospital Universitario 12 de Octubre, Madrid, Spain, doi: 10.3389/fped.2020.614521 31 Department of Pediatric Hemato-Oncology and Stem Cell Transplantation, La Paz University Hospital, Madrid, Spain, Frontiers in Pediatrics | www.frontiersin.org 1 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL Pediatric Onco-Hematology Department, Hospital Universitario Donostia, Donostia, Spain, 33 Pediatric Onco-Hematology 32 Department, Complejo Hospitalario de Navarra, Navarra, Spain, 34 Paediatric Hematology Department, Hospital Parc Taulí, Sabadell, Spain, 35 Haematology and Oncology Department, Hospital de Cruces, Bilbao, Spain Robust and applicable risk-stratifying genetic factors at diagnosis in pediatric T-cell acute lymphoblastic leukemia (T-ALL) are still lacking, and most protocols rely on measurable residual disease (MRD) assessment. In our study, we aimed to analyze the impact of NOTCH1, FBXW7, PTEN, and RAS mutations, the measurable residual disease (MRD) levels assessed by flow cytometry (FCM-MRD) and other reported risk factors in a Spanish cohort of pediatric T-ALL patients. We included 199 patients treated with SEHOP and PETHEMA consecutive protocols from 1998 to 2019. We observed a better outcome of patients included in the newest SEHOP-PETHEMA-2013 protocol compared to the previous SHOP-2005 cohort. FCM-MRD significantly predicted outcome in both protocols, but the impact at early and late time points differed between protocols. The impact of FCM-MRD at late time points was more evident in SEHOP-PETHEMA 2013, whereas in SHOP-2005 FCM-MRD was predictive of outcome at early time points. Genetics impact was different in SHOP-2005 and SEHOP-PETHEMA-2013 cohorts: NOTCH1 mutations impacted on overall survival only in the SEHOP-PETHEMA-2013 cohort, whereas homozygous deletions of CDKN2A/B had a significantly higher CIR in SHOP-2005 patients. We applied the clinical classification combining oncogenetics, WBC count and MRD levels at the end of induction as previously reported by the FRALLE group. Using this score, we identified different subgroups of patients with statistically different outcome in both Spanish cohorts. In SHOP-2005, the FRALLE classifier identified a subgroup of high-risk patients with poorer survival. In the newest protocol SEHOP-PETHEMA-2013, a very low-risk group of patients with excellent outcome and no relapses was detected, with borderline significance. Overall, FCM-MRD, WBC count and oncogenetics may refine the risk-stratification, helping to design tailored approaches for pediatric T-ALL patients. Keywords: measurable (minimal) residual disease, T-cell acute lymphoblastic leukemia, oncogenetics, NOTCH1, flow cytometry, pediatrics, risk-factors INTRODUCTION quantification post-remission remains the most important risk factor (3–5). T-ALL patients show a slower blast clearance Compared to the particularly good outcome of pediatric patients compared to BCP-ALL, and the MRD detection at a late time with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), point (day 78) may define better the risk of relapse in T-ALL T-ALL patients still do worse, and 15–20% of pediatric and patients than earlier evaluation at end of induction (day 33) 40% of adult T-ALL patients relapse. Importantly, relapsed T- in some therapeutic protocols (6). The MRD gold standard ALL is often highly resistant, and such patients present a dismal method is real-time quantitative polymerase chain reaction for prognosis (1, 2). Hence, prevention of relapse is imperative, and immunoglobulin and T-cell receptor clonality (IG-TR PCR- patients are allocated to intensive and often very toxic therapeutic MRD) (6–8). Flow-cytometry MRD measurement (FCM-MRD), regimes with short- and long-term side effects. A precise risk based on the leukemia-associated immunophenotype, is also used stratification of T-ALL patients would allow to dynamically for risk stratification in some collaborative groups and provides adjust the intensity of treatment, helping to balance the risks reliable identification of patients eligible for reduced-intensity of relapse and treatment-related toxicity. Current therapeutic therapy in T-ALL and BCP-ALL (9–12). However, even in the protocols aim to tailor treatment by applying less intensive setting of MRD-oriented protocols, some T-ALL patients relapse, therapy to low-risk patients, thus reducing the risk of treatment- and it is important to identify additional prognostic factors. related toxicity, while reserving intensive therapy to high-risk Multiple genetic abnormalities have been identified in T- patients. In contrast to BCP-ALL, the new biological insights in ALL cases (13, 14), including proto-oncogene activation, tumor- T-ALL have scarcely been incorporated to current protocols, and suppressor gene deletions and constitutive activation of the risk stratification relies mainly on response to treatment. Thus, NOTCH1 pathway, present in 40–60% of patients (15–17). although white blood cell (WBC) count and early response to The prognostic impact of NOTCH1 and FBXW7 mutations prednisone at day 8 are used, measurable residual disease (MRD) is controversial; it has been usually associated with favorable Frontiers in Pediatrics | www.frontiersin.org 2 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL prognosis (18–20), but other studies have found no significant diagnosed with T-ALL in Spain with available biological material. effect (21, 22), and some authors have reported that mutant The participation in the study was offered to all the Spanish FBXW7 independently predicts an inferior survival (23). Recent centers belonging to the SEHOP Group of Leukemias. DNA studies indicate that the impact of NOTCH1/FBXW7 mutations samples from 26 different Spanish centers were sent to Hospital could be modulated by concomitant mutations in PTEN and Sant Joan de Déu to perform the oncogenetic studies. The RAS genes (3). PTEN loss through mutation or genomic deletion referring centers provided data about clinical and basic biological occurs in up to 35% of T-ALL pediatric patients, and ∼10% characterization of their patients at diagnosis and the levels harbor N- or K-RAS mutations. Although these abnormalities of MRD during follow-up. The clinic-biological data of the are usually associated with unfavorable prognosis, their impact patients included in our study are detailed in Table 1 and in pediatric T-ALL patients is still unclear (24–27). The Group Supplementary Table 1. A total of 199 pediatric T-ALL patients for Research in Adult Acute Lymphoblastic Leukemia (GRAALL) treated according to SEHOP and PETHEMA Cooperative validated in adult T-ALL patients the clinical utility of a Groups from 1998 to 2019 were collected. We analyzed the new oncogenetic classifier based on the mutational status of molecular features of 189 patients, after excluding cases with NOTCH1/FBXW7/PTEN/RAS (NFPR) genes (28). In this model, T-cell lymphoblastic lymphoma, and patients with insufficient the presence of NOTCH1 and FBXW7 mutations without lesions sample or no clinical data. For the survival analysis, we excluded involving PTEN/RAS defined the genetic low-risk subgroup those patients treated as per PETHEMA protocols, as they had (gLoR), and other genotypes were allocated to the genetic high- been treated heterogeneously according to different therapeutic risk group (gHiR). In contrast, in a study in 145 pediatric T-ALL regimes. The 142 patients selected for the survival analyses were patients treated with the MRC UKALL2003 trial, neither PTEN treated according to the consecutive protocols SHOP-2005 (n = nor RAS significantly impacted on outcome, and none of these 51) and SEHOP-PETHEMA-2013 (n = 91). These patients were mutations changed the highly favorable outcome of patients with representative of the whole SHOP-2005 and SEHOP-PETHEMA- double NOTCH1/FBXW7 mutations (29). Recently, the FRALLE 2013 population in terms of clinical characteristics at diagnosis group (French Acute Lymphoblastic Leukemia Study Group) and outcome and only differed in a longer follow-up for the suggested a new classifier based on WBC count, MRD by IG- patients included in the study (Supplementary Table 2). TR PCR-MRD and the oncogenetic mutational status, which SHOP-2005 (2005–2013) and SEHOP-PETHEMA-2013 improved prediction of the relapse risk for their pediatric T-ALL (2013-present) are two consecutive therapeutic protocols patients (3). BFM (Berlin-Frankfurt-Münster)-inspired. The former Copy number alterations (CNA) have been reported as has been previously described (5), and the full details prognostic markers, both in BCP-ALL and in T-ALL (30, 31). of SEHOP-PETHEMA-2013 protocol are provided in the CDKN2A/B loci deletion is present in up to 70% of T-ALL Supplementary Material. Patients with T-ALL were not eligible patients. In adult T-ALL, CDKN2A/B homozygous deletion for standard risk group stratification in either protocol. To (CDKN2A/Bhomo ) was associated with a favorable prognosis only summarize the main differences between both protocols, in the within the mature/cortical group (32), while other studies showed newer SEHOP-PETHEMA-2013: (1) prednisone response was a correlation with poor survival or no association with outcome incorporated as initial stratification criteria; (2) an Induction IB (31, 33). Overall, the role of oncogenetics as risk factors in course was added; (3) craniospinal irradiation was omitted and T-ALL is controversial and seems to differ according to the increased number of doses of triple intrathecal therapy were used treatment protocol. instead; (4) there was a more intensive use of asparaginase, with We present a retrospective collaborative study of the more doses of asparaginase, use of PEG-asparaginase and longer Biological Committee of the Leukemia Group of the SEHOP periods of asparagine depletion; (5) there were more restrictive (Spanish Society of Pediatric Hematology and Oncology) on indications of allogeneic stem cell transplantation (allo-SCT), a series of 199 pediatric T-ALL patients treated according mainly based on FCM-MRD criteria, and (6) eight-colors FCM- to SEHOP and PETHEMA (Programa para el Estudio de la MRD was performed in reference laboratories, in contrast with Terapéutica en Hemopatías Malignas) Cooperative Groups. In decentralized 4-color FCM-MRD in SHOP-2005 (see below). both protocols, we aimed to: (1) confirm the clinical impact of FCM-MRD measurement, (2) assess the prognostic significance of MRD at late time points, and (3) test whether the previously FCM Characterization and MRD reported oncogenetic classifier by the FRALLE group could Assessment refine the risk stratification in our patients receiving BFM- The immunophenotyping by FCM was performed in each based protocols. laboratory using the combination of antibodies and panels detailed for each protocol in the Supplementary Material. Briefly, in SHOP-2005 protocol, 4-color panels were used in each MATERIALS AND METHODS center for identification and monitoring of leukemia-associated immunophenotypes (LAIPs), according to the guidelines of Study Design, Cohort of Patients, and group EGIL (34). For residual disease analysis, either patient- Therapeutic Protocols tailored antibody combinations or those employed at diagnosis The study design is shown in Supplementary Figure 1. This were used. In this protocol, MRD levels >0.1% were considered is a retrospective study, designed to include pediatric patients positive and used for clinical decisions. However, lower values Frontiers in Pediatrics | www.frontiersin.org 3 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL TABLE 1 | Clinical and molecular features of patients included in a required resolution of at least 20 events to refer a sample as SEHOP-PETHEMA-2013 and SHOP-2005 protocols for the survival analyses. positive), an optimum of 500,000 nucleated cells needed to be SHOP-2005 SEHOP-PETHEMA-2013 p-value acquired. MRD was assessed at day 15, end of induction (time (n = 51) (n = 91) point 1, TP1) and after the second course of chemotherapy, around day 78 (timepoint 2, TP2). In this protocol, MRD levels Sex n = 51 n = 91 >0.01% were considered positive, and the thresholds considered Male 38 (74.5%) 67 (73.6%) 1.00 for clinical decisions varied according to the timepoint. Thus, Female 13 (25.5%) 24 (26.4%) patients were stratified to high-risk arm if FCM-MRD levels were Age, years, median 8.72 [1.69; 19.0] 7.49 [1.43; 16.0] 0.30 >10% at day 15, >1% at the end of induction (TP1) and >0.1% at [range min; max] the end of consolidation (TP2). Patients were allocated to receive Age n = 51 n = 91 0.37 an allo-SCT in case of: (1) not complete remission (CR) after TP1; <10 years 29 (56.9%) 60 (65.9%) (2) day 33 FCM-MRD level >1%, and FCM-MRD level >0.1% ≥10 years 22 (43.1%) 31 (34.1%) at TP2; or (3) persistence of positive FCM-MRD (>0.01%) after WBC count, median 56.9 [1.00; 675] 72.2 [1.90; 897] 0.51 high-risk blocks of chemotherapy. [range min; max] Globally, irrespectively, of the protocol treatment, we used a WBC count n = 51 n = 89 0.88 sensitivity threshold of 0.01% in this study to try to reproduce <200 × 109 /L 37 (72.5%) 67 (75.3%) the previously reported results in the FRALLE and GRAALL protocols (3, 28). ≥200 × 109 /L 14 (27.5%) 22 (24.7%) CNS n = 51 n = 83 1 47 (92.2%) 55 (66.3%) Mutation Screening and CNA Analysis 2 4 (7.84%) 12 (14.5%) 0.001 We analyzed the hotspot regions reported for NOTCH1 (exons 3 0 (0.00%) 16 (19.3%) 26, 27, 28, 34), FBXW7 (exons 9 and 10), PTEN (exon 7), NRAS Phenotype n = 37 n = 83 (exons 1 and 2), and KRAS (exons 1 and 2) by Sanger sequencing Cortical 21 (56.8%) 41 (49.4%) 0.58 as previously described (25, 36). CNAs were screened by MLPA using the SALSA MLPA P383 T-ALL kit (MRC Holland, Other 16 (43.2%) 42 (50.6%) Amsterdam, The Netherlands), according to the manufacturer’s NOTCH1 n = 47 n = 90 instructions. We used the Coffalyser software v.140721.1958 for Wild type 32 (68.1%) 57 (63.3%) 0.72 the analysis (MRC-Holland, Amsterdam, The Netherlands). The Mutated 15 (31.9%) 33 (36.7%) P383 T-ALL kit assesses alterations (deletions or amplifications) FBXW7 n = 51 n = 89 in transcription factors (LEF1 and MYB), genes involved in Wild type 42 (82.4%) 77 (86.5%) 0.68 signal transduction (PTEN, NF1, and PTPN2), in cell cycle Mutated 9 (17.6%) 12 (13.5%) (CDKN2A, CDKN2B, and CASP8AP2), in epigenetic regulation PTEN n = 51 n = 89 (EZH2, SUZ12, and PHF6), and identifies the STIL-TAL1 and Wild type 46 (90.2%) 79 (88.8%) 1.00 NUP214-ABL1 fusion genes. Full experimental details are given in the Supplementary Methods. Abnormality 5 (9.80%) 10 (11.2%) N/K-RAS n = 51 n = 89 Wild type 45 (88.2%) 84 (94.4%) 0.21 Outcome Analyses Mutated 6 (11.8%) 5 (5.62%) To assess the predictive value of the conventional and new Oncogenetics n = 44 n = 82 oncogenetic risk factors, we analyzed the prognostic impact of the gLoR 15 (34.1%) 31 (37.8%) 0.83 NFPR mutational status, first addressing each gene individually, and then grouped as previously reported (20). We applied the gHiR 29 (65.9%) 51 (62.2%) GRAALL oncogenetic classifier (28), and grouped our patients Fusion-gene n = 41 n = 81 into gLoR and gHiR to compare their outcome. We also analyzed NUP214-ABL1 3 (7.32%) 2 (2.47%) 0.23 our patients with the FRALLE score, combining the oncogenetic STIL-TAL1 6 (14.6%) 12 (14.8%) 0.36 classifier by GRAALL group with WBC count ≥200 × 109 /L and WBC, white blood cell; CNS, central nervous system; meaning 1: no blasts in CNS, 2: the MRD levels at TP1 (3), plus an additional time point at TP2. blasts in CNS with ≤5 WBC/µl and/or traumatic lumbar puncture, and 3: blast in CNS with <5 WBC/µl or CNS symptoms. Statistical Analysis Statistical analyses were carried out using R software (37), considering all p-values lower than 0.05 to be statistically were reviewed and recorded for the analysis in the present significant. Details are provided in Supporting Information. study. In SEHOP-PETHEMA-2013 protocol, an 8-colors panel according to EuroFlow consortium was recommended at diagnosis (35), and FCM-MRD was centralized in 10 reference Ethical Aspects laboratories. For MRD detection down to the 0.01% level (with The ethical issues are detailed in Supplementary Material. Frontiers in Pediatrics | www.frontiersin.org 4 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL RESULTS FCM-MRD Predicted Outcome in Molecular Findings and Association With Consecutive SEHOP Protocols FCM-MRD data in the SHOP-2005 cohort was available for 46 Clinical Features at Diagnosis and 35 out of 51 patients at TP1 and TP2, respectively. At TP1, Molecular findings are shown in Table 1 and patients with MRD < 0.01% (n = 33) showed a significantly Supplementary Figure 2. Sixty-four patients (35%) harbored better outcome than patients with MRD ≥ 0.01%, with a 5-year NOTCH1 mutations (NOTCH1mut ), 45% at HD-N domain, OS of 84.8 vs. 53.8%, a 5-year DFS of 78.7 vs. 46.2%, and a CIR of 5% at HD-C domain and 9% at PEST domain. No mutations 12.6 vs. 38.5% (p = 0.019, p = 0.009, and p = 0.039, respectively). were found at TAD domain (Supplementary Figure 3). Out of At TP2, with less data available, we observed only a borderline NOTCH1mut patients, eleven cases with HD-C mutations significant difference with worse OS in patients with FCM-MRD also had concomitant mutations at the PEST domain ≥ 0.01% (p = 0.050; Supplementary Figure 6). (NOTCH1double ). FBXW7 was mutated in 25 cases (13%), In SEHOP-PETHEMA-2013 patients, FCM-MRD data was alone in 16 patients and combined with NOTCH1mut in 9 available in 84 and 62 out of 91 cases at TP1 and TP2, cases. Overall, we observed NOTCH1/FBXW7 mutations in respectively. In contrast with the former protocol, FCM-MRD 80/182 cases (44%), in line with previous reports, albeit in significantly impacted on outcome at TP2 (5-year DFS for the lower range. PTEN abnormalities (mutation or major MRD <0.01% 92.6 vs. 68.6% for MRD ≥ 0.01%, p = 0.026; deletion, PTEN abn ) were found in 30 patients (20%), including Supplementary Figure 7), whereas at TP1 only impacted on OS 9 with NOTCH1/FBXW7 mutations. K/N-RAS mutations were with a borderline significance (p = 0.049). identified in 14 patients (8%). Overall, 108 patients (57%) harbored at least one mutation/deletion in NOTCH1, FBXW7, PTEN, or K/N-RAS. The mutations included point mutations, Oncogenetics Impact on Outcome insertions, deletions and indels (Supplementary Table 3 and In SHOP-2005 cohort, NOTCH1 mutations did not significantly Supplementary Figure 3). CDKN2A and CDKN2B were deleted impact on outcome. Of note, all patients with NOTCH1double in 109 out of the 160 tested cases (68%) and 95 patients (60%), mutations (n = 3) were alive in continuous CR (data not respectively, including 89 homozygous deletions for both genes. shown). Using the combination of the NFPR mutational status The association of NOTCH1, FBXW7, PTEN abn , and K/N-RAS defined in the GRAAL oncogenetic stratifier (gLoR vs. gHiR), status with clinical variables is shown in Supplementary Table 4. we observed no differences in patients’outcome (Figure 1A). In Globally, and in each protocol, NOTCH1 mutations significantly contrast, patients with CDKN2A/Bhomo had a trend toward a associated with females, while K-RAS mutations associated with worse DFS (p = 0.079) and a significantly higher CIR than WBC count ≥200 × 109 /L. patients with normal or heterozygous deletions of CDKN2A/B (p We could classify 168 patients according to the previously = 0.009, Supplementary Figure 8a). reported oncogenetic stratifier (Table 1). Fifty-eight patients In patients treated with SEHOP-PETHEMA-2013 protocol, (34.5%) presented NOTCH1/FBXW7 mutations without a those cases with NOTCH1mut showed a better OS (p = 0.045), PTEN/RAS mutation (NFmut PRwt ) and were considered as and all patients with NOTCH1double mutations (n = 7) were alive gLoR; the remaining 110 cases (65.5%) were classified as in continuous CR (data not shown). In contrast to the former gHiR. The gHiR group included 15 positive patients for protocol, those SEHOP-PETHEMA-2013 patients classified as both NOTCH1/FBXW7 and PTEN/RAS mutations, 26 patients gLoR presented a significantly better DFS (p = 0.043), and a trend harboring PTEN/RAS mutations only and 69 patients with all toward a better OS and a lower CIR (Figure 1B). Patients with genes in a wild-type status. The distribution of oncogenetic CDKN2A/Bhomo had a worse outcome, but the difference was not risk-groups between SHOP-2005 and SEHOP-PETHEMA-2013 statistically significant (Supplementary Figure 8b). cohorts was similar (Table 1). Overall, we observed a different impact of oncogenetics on the SHOP-2005 and SEHOP-PETHEMA-2013 series of patients. Patients’ Outcome in Consecutive SEHOP Prognostic Impact of Classical Risk Protocols Factors The outcome of the 142 assessable patients according to the The univariate and multivariate analyses of OS and DFS for therapeutic protocol is shown in Supplementary Figures 4, 5. patients in SHOP-2005 and SEHOP-PETHEMA-2013 protocols The median follow-up of the patients was 7.7 years for SHOP- is shown in Table 2. FCM-MRD was significantly predictive 2005 and 2.8 years for SEHOP-PETHEMA-2013 cohort. Overall, of outcome in both SHOP-2005 and SEHOP-PETHEMA- we observed a non-significantly better outcome in the newest 2013 cohorts (Supplementary Figures 6, 7), being statistically protocol: the 5-year overall survival (OS) was 85.9 ± 4.0 vs. significant at early TP1 only for SHOP-2005 (p = 0.035) and 76.5 ± 5.9% (p = 0.22), and the disease-free survival (DFS) was at late TP2 only for SEHOP-PETHEMA 2013 protocol (p = 85.3 ± 4.1 vs. 70.5 ± 6.4% (p = 0.080) for SEHOP-PETHEMA- 0.048). However, other classical risk factors impacted differently 2013 and SHOP-2005 patients, respectively. Noticeably, the depending on the protocol: patients with hyperleukocytosis ≥200 cumulative incidence of relapse (CIR) was not statistically × 109 /L showed a significantly worse DFS only in SHOP-2005 different in both protocols (12.1 ± 3.8% vs. 18.1 ± 5.5%, series. Sex, age, and CNS infiltration did not impact on outcome p = 0.49). in either protocol. Frontiers in Pediatrics | www.frontiersin.org 5 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL FIGURE 1 | Survival of patients included in SHOP-2005 and SEHOP-PETHEMA-2013 protocol according to oncogenetics. (A) OS, DFS, and CIR in patients included in the SHOP-2005 protocol with gLoR vs. gHiR; (B) OS, DFS, and CIR in patients included in the SEHOP-PETHEMA-2013 protocol with gLoR vs. gHiR. gLoR: presence of NOTCH1 and FBXW7 mutations without lesions involving PTEN/RAS; gHiR: all the other genotypes. Use of a Clinical Prognostic Scores the FRALLE group, impacted significantly on outcome, and Combining Classical and New Risk Factors identified different risk populations in the analyzed cohorts. We used different combinations of classical and new risk factors to gain additional information on the relapse risk. First, we DISCUSSION wanted to test in our series of patients the clinical classification reported by Petit et al. (3) integrating oncogenetics, MRD at Most pediatric T-ALL protocols only rely on MRD assessment TP1 (threshold 0.01%) and WBC count. By doing that, we to guide stratification in risk-groups, and robust and applicable observed a different distribution of subgroups in the analysis risk-stratifying genetic factors at diagnosis are still lacking. by protocols: in SHOP-2005, the outcome analysis following the The FRALLE group built a classifier based on WBC count, clinical classification defined by the FRALLE group showed a MRD at TP1 and an oncogenetic stratifier according to NFPR poorer survival of high-risk patients as compared to the similar mutational status. However, some questions remain open outcome of the low- and intermediate-risk patients (OS p = 0.25, regarding the FRALLE classifier: (1) would it be predictive of DFS p = 0.024, and CIR p = 0.044; data not shown). In contrast, outcome in patients treated with BFM-based pediatric protocols?; in SEHOP-PETHEMA-2013 patients, the low-risk patients had (2) could MRD at TP2 be applied?, and (3) would this a better not significant outcome as compared to the survival of classifier be similarly predictive of patients’ outcome when intermediate- and high-risk groups (p = 0.15, data not shown). assessing MRD by FCM? To answer these questions, we We grouped the latter groups with similar outcome, and found analyzed a large cohort of pediatric T-ALL patients treated that the clinical classifier was able to identify with borderline with consecutive Spanish protocols. We confirmed MRD as a significance low-risk patients with 100% OS and no relapses (p = prognostic risk-factor throughout different protocols, also when 0.059, p = 0.05, and p = 0.06 for OS, DFS, and CIR, respectively) measured by flow-cytometry. However, oncogenetics impacted (Figure 2). differently according to treatment, confirming that therapeutic Taken together, the oncogenetics, FCM-MRD at TP1 and modifications can lead to changes in the prognostic impact of WBC count, combined as defined by Petit et al. (3) in biological variables like NOTCH1 mutations. Frontiers in Pediatrics | www.frontiersin.org 6 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL TABLE 2 | Univariate and multivariate analysis of OS and DFS for patients in SHOP-2005 and SEHOP-PETHEMA-2013 protocols. SHOP-2005 protocol SEHOP-PETHEMA-2013 protocol Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis HR [95% CI] p HR [95% CI] p HR [95% CI] p HR [95% CI] p OVERALL SURVIVAL (OS) Age, years 0.99 [0.87; 1.12] 0.83 – – 1.03 [0.89; 1.19] 0.67 – – Sex Female Ref Ref – – Ref Ref – – Male 4.73 [0.61; 36.39] 0.14 – – 1.71 [0.37; 7.90] 0.49 – – WBC count <200 × 109 /L Ref Ref – – Ref Ref – – ≥200 × 109 /L 2.00 [0.65; 6.11] 0.23 – – 1.22 [0.32; 4.62] 0.77 – – CNS involvement Yes Ref Ref – – Ref Ref – – No 0.38 [0.00; 2.41] 0.44 – – 1.50 [0.42; 5.33] 0.53 – – MRD TP1 <0.01% Ref Ref – – Ref Ref – – ≥0.01% 3.56 [1.14; 11.11] 0.028 – – 3.51 [0.93; 13.29] 0.064 – – MRD TP2 <0.01% Ref Ref – – Ref Ref – – ≥0.01% 3.80 [0.90; 16.00] 0.069 – – 4.40 [0.80; 24.15] 0.088 – – Oncogenetics gLoR Ref Ref – – Ref Ref – – gHiR 1.17 [0.30; 4.51] 0.82 – – 5.73 [0.73; 45.27] 0.098 – – DISEASE FREE SURVIVAL (DFS) Age, Years 0.95 [0.84; 1.07] 0.38 – – 1.09 [0.94; 1.26] 0.26 – – Sex Female Ref Ref – – Ref Ref – – Male 5.89 [0.77; 44.82] 0.087 1.66 [0.36; 7.69] 0.52 WBC count <200 × 109 /L Ref Ref Ref Ref Ref Ref – – ≥200 × 109 /L 2.88 [1.04; 7.96] 0.042 3.25 [1.12; 9.47] 0.031 1.33 [0.35; 5.02] 0.67 CNS involvement Yes Ref Ref – – Ref Ref – – No 0.33 [0.00; 2.41] 0.35 – – 0.99 [0.26; 3.83] 0.99 – – MRD TP1 <0.01% Ref Ref Ref Ref Ref Ref – – ≥0.01% 3.67 [1.28; 10.50] 0.016 3.15 [1.09; 9.18] 0.035 1.11 [0.29; 4.29] 0.88 – – MRD TP2 <0.01% Ref Ref – – Ref Ref Ref Ref ≥0.01% 3.04 [0.76; 12.21] 0.12 – – 5.59 [1.02; 30.70] 0.048 5.59 [1.02; 30.70] 0.048 Oncogenetics gLoR Ref Ref – – Ref Ref – – gHiR 0.98 [0.29; 3.24] 0.97 – – 6.37 [0.81; 49.76] 0.078 – – HR, hazard ratio; CI, confidence interval; Ref, reference category; WBC, white blood cell; CNS, central nervous system; MRD, measurable residual disease; TP1, end of induction; TP2, end of consolidation; gLoR, genetic low-risk according to the oncogenetic stratifier; gHiR, genetic high-risk according to the oncogenetic stratifier. Bold values were considered significative as p < 0.05. We first evaluated the outcome of Spanish pediatric could partly explain the different outcome. Briefly, both are T-ALL patients in consecutive protocols. We observed a not BFM-inspired protocols, but in SEHOP-PETHEMA-2013 statistically significant improvement in the 5-year DFS with an induction IB course with cyclophosphamide, cytarabine, SEHOP-PETHEMA-2013 protocol, as compared with the older and mercaptopurine was incorporated. In this regard, an SHOP-2005 protocol. The differences between both protocols intensive induction and multiagent consolidation including Frontiers in Pediatrics | www.frontiersin.org 7 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL FIGURE 2 | Outcome of patients included in SEHOP-PETHEMA-2013 according to the clinical classifier defined by Petit et al. (3). OS, DFS, and CIR in SEHOP-PETHEMA-2013 patients according to the FRALLE group combination, comparing the outcome of low-risk patients with the intermediate-risk and high-risk pooled together. The clinical classifier identified a subgroup of low-risk patients with excellent outcome, 100% OS at 5 years and no relapses. We observed a trend to the statistical significance in the outcome of the low-risk group vs. the remaining patients. cyclophosphamide is strongly recommended in T-ALL (38), and methods (11). In our study, FCM-MRD discriminated outcome omitting the induction IB resulted in inferior outcome in large both at early and late time points in SHOP-2005, whereas reported cohorts (39, 40). Also, a more intense asparaginase significant differences were observed in SEHOP-PETHEMA- scheme including PEG-asparaginase was given in SEHOP- 2013 cohort at TP2. This could be driven by differences PETHEMA-2013. Thus, as compared to SHOP-2005 protocol, between both protocols in terms of intensity of treatment, with patients received increased doses of asparaginase and longer earlier stratification criteria using prednisone response, and early periods of asparagine depletion. The response to treatment intensification therapy being added in SEHOP-PETHEMA-2013 was also differently assessed: in SEHOP-PETHEMA-2013 patients, mimicking the backbone of current BFM protocols. the prednisone response was added as stratifying criteria, Importantly, methodological differences in the FCM-MRD and the non-centralized 4-colors FCM-MRD was replaced assessment between SHOP-2005 and SEHOP-PETHEMA-2013 by 8-colors FCM-MRD with agreed panels, centralized can play a role in the results. Thus, in the latter protocol, 8-color in reference laboratories. Moreover, a lower number of FCM-MRD centralized in reference laboratories, replaced the patients underwent an allo-SCT in SEHOP-PETHEMA-2013 non-centralized 4-color FCM-MRD performed in SHOP-2005. (Supplementary Figure 4), and had a lower non-leukemic The increased number of markers in SEHOP-PETHEMA-2013 mortality (data not shown). Overall, the intensification of the should, theoretically, improve the accuracy of the analysis, but chemotherapy, a better stratification of patients and lower non- we did not investigate the concordance between FCM and other leukemic mortality in the latter protocol, could have contributed methods like IG-TR MRD. On the other hand, the sensitivity to the improvement of the survival in SEHOP-PETHEMA-2013 in the FCM studies performed in SEHOP-PETHEMA-2013 was cohort. Similarly, a survival benefit dependent on mainly expected to be higher than in SHOP-2005 due to a higher number reducing toxicity alongside improving efficacy was recently of acquired cells, but this point has not been confirmed nor described in adult T-ALL patients (41). a straightforward comparison between methodologies has been We then assessed the prognostic impact of conventional and performed. Globally, in the SEHOP-PETHEMA-2013 cohort, new risk factors within each protocol. Hyperleukocytosis ≥200 × FCM-MRD reproduced previous remarks about the relevance of 109 /L was only predictive of a significantly worse DFS in SHOP- late time points in BFM-based protocols. Overall, although the 2005 cohort. Next, we analyzed the prognostic value of FCM- number of patients in each protocol is low, and even considering MRD in our cohorts. FCM-MRD levels impacted significantly on the less controlled setting in the older protocols, FCM-MRD patients’ outcome in both SHOP-2005 and SEHOP-PETHEMA- appeared as a robust risk-factor in our Spanish series of patients. 2013 protocols, proving the usefulness of FCM-MRD with a As reported, we confirmed in our cohorts recurrent threshold of 0.01% to discriminate prognosis. This is especially mutations in pediatric T-ALL patients. We found alterations in relevant in the difficult setting of flow-cytometry assessment NOTCH1/FBXW7 genes in 44% of our patients. The incidence of MRD in T-ALL patients. However, FCM-MRD impacted of NOTCH1 mutated cases in our series is low. However, similar differently according to the protocol and time point. The impact incidences have been published in other series, ranging from of MRD at late time points in T-ALL patients may vary between 23 to 70.8% (42, 43). Specifically, 23 and 40% of NOTCH1 protocols, as reported by AIEOP-BFM and NOPHO Groups mutations were reported in Turkish (44) and Japanese cohorts (6, 11). The discrepancies could be explained by differences in of patients (45), respectively. FBXW7 mutations also range from the treatment protocols, stratification criteria, cutoff levels and 8.6 to 30.8% in the literature. Overall, the frequency of NFPR Frontiers in Pediatrics | www.frontiersin.org 8 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL mutations, and mutation rates of CDKN2A/B deletions, were in and no de-intensification is considered. Interestingly, following the range of previously reported incidences (42, 46–48). When the FRALLE risk stratification, a quarter of our intermediate- we classified patients into gLoR and gHiR, patients were grouped risk patients (20/76, 26%) could be considered as candidates in the same way in both protocols. However, we found a lower to receive less intense treatment, and be moved to a lower- proportion of gLoR patients compared to the FRALLE group, in risk group. Our results, if confirmed in larger series, could help parallel to the lower incidence of NOTCH1/FBXW7 mutations in to consider the option of selected de-intensification therapy in our cohort. The highly variable frequency in NOTCH1/FBXW7 future clinical trials. mutations could be explained by different factors including bias Some factors may limit our study. Centralization in reference in sample storage, ethnicity, methodological aspects, and the total laboratories and biobanking was not mandatory in former number of patients. protocols. Also, to assess the possible impact of differences in the Regarding oncogenetic impact, we first studied the impact therapeutic regimes, we analyzed the outcome of each protocol of individual genetic alterations. NOTCH1 mutations had a separately. Hence, the low number of patients in each group may favorable clinical impact in SEHOP-PETHEMA-2013 cohort, have limited the power of statistics and precluded the finding and all patients with NOTCH1double mutations were alive in of significant results. Finally, the median follow-up of SEHOP- continuous complete remission. Neither PTEN nor K/N-RAS PETHEMA-2013 cohort is short (2.8 years), but it covers most of mutations had a significant impact on outcome. As reported, the the expected T-ALL relapses. homozygous deletion of CDKN2A/B associated with higher CIR In summary, we present a national collaborative study of in SHOP-2005 patients. In this line, recent studies have shown clinical and genetic prognostic factors in a large series of T-ALL that the lack of deletions involving CDKN2A/ARF/CDKN2B pediatric patients. Notably, FCM-MRD predicted outcome in locus, combined with undetectable MRD (≤0.01%) values, both Spanish protocols, being more important in TP2 in SEHOP- allowed the identification of a subset of adult T-ALL patients with PETHEMA-2013. We could reproduce the FRALLE group’s better OS in the absence of allo-SCT (31). results in our BFM-based SEHOP protocols, and observed the The analysis of the combination of oncogenetic abnormalities predictive value of the combination of FCM-MRD, WBC count yielded different results according to the protocols. Hence, and oncogenetics to predict outcome at TP1. This classifier the application of the oncogenetic classifier (NFPR) to our allowed, in our current protocol SEHOP-PETHEMA-2013, to cohorts showed no impact in SHOP-2005 patients and a identify a low-risk subgroup of patients with excellent outcome. trend to a better OS and CIR, and a significantly better Further studies in the context of controlled clinical trials would be DFS in SEHOP-PETHEMA-2013 patients. We next tested the necessary to confirm if the de-intensification therapy of a selected clinical classification reported by the FRALLE group, integrating low-risk group of patients would lead to the same good outcome. oncogenetics, MRD at TP1 (threshold 0.01%) and WBC count. Our results provide data that could be clinically relevant, as may The FRALLE classifier allowed to identify different subgroups help to apply tailored risk-directed treatments to reduce both of patients with statistically different outcome in the whole toxicity and relapse. cohort of Spanish patients. In the newest protocol SEHOP- PETHEMA-2013, a very low-risk group could be segregated with this score. Thereby, we reproduced the results of the DATA AVAILABILITY STATEMENT FRALLE group in different, BFM-inspired therapeutic protocols. However, the results in SHOP-2005 and SEHOP-PETHEMA- The original contributions presented in the study are included 2013 analyzed individually varied, probably due to differences in the article/Supplementary Material, further inquiries can be between protocols. directed to the corresponding author/s. The FRALLE-2000 protocol differed in several aspects from SHOP-2005 and SEHOP-PETHEMA-2013. Briefly, FRALLE- 2000 and SHOP-2005 had similar indications of cranial ETHICS STATEMENT irradiation, given both prophylactically in a subgroup of The studies involving human participants were reviewed and patients and therapeutically in all T-ALL patients, whereas approved by Comitè Ètic d’Investigació amb medicaments cranial irradiation was omitted in SEHOP-PETHEMA-2013. (CEIm) de la Fundació Sant Joan de Déu. Written informed The FRALLE-2000 protocol had more cyclophosphamide and consent to participate in this study was provided by the anthracycline cumulative doses than SHOP-2005. On the other participants’ legal guardian/next of kin. hand, SEHOP-PETHEMA-2013 had a prolonged intensification with PEG-asparaginase compared with FRALLE-2000 and SHOP-2005 protocols. Taken together, we observed that, even AUTHOR CONTRIBUTIONS in the setting of different protocols, the oncogenetics, FCM- MRD at TP1, and WBC count, may be useful to refine the NV-G and MC designed the study and MC supervised the risk-stratification in pediatric T-ALL patients. In BFM-based project. NV-G, EE-C, and CV-G performed the molecular protocols, the FRALLE classifier allowed to identify a subgroup analyses of the study. NV-G and SP-J analyzed the data of patients with very low-risk, who could be considered as and performed the statistical analyses. NV-G and MC wrote potential candidates to de-intensify treatment. Our current the paper with the contribution of SP-J, MT, JD, and SR. protocol, SEHOP-PETHEMA-2013, aims to intensify treatment, MT, AJ-V, MO, ML, LA, JV, AM, MP, JS, CG-C, MG-C, Frontiers in Pediatrics | www.frontiersin.org 9 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL EM-C, AE, MM-G, LM, MV, MG-M and MC diagnosed PARTICIPANT INVESTIGATORS patients and performed the main biological characterization of samples. AC, AP, AJ-V, JMF, AL, JLF, IB, AMo, AM-Q, The authors thank the contribution of other clinicians and PG-G, APM, MG, RR, SP, RA, JD, SR and MR-O recruited researchers from different centers integrated in the Group of patients. All authors read and approved the final version of Leukemia of the Spanish Society of Pediatric Hematology and the manuscript. Other centers and investigators belonging to Oncology (SEHOP), for collecting data, providing and caring the Biological Committee and other integrands of the Group of for study patients: Hospital Sant Joan de Déu (Nuria Conde, Leukemia of the Spanish Society of Pediatric Hematology and Sandra Pont, Sara Montesdeoca, Ignacio Isola); Hospital Niño Oncology (SEHOP) contributing to this study are listed in the Jesús (Ana Castillo), Hospital Universitari i Politècnic La Fe (Inés participant investigators. Gomez-Seguí), Hospital Carlos Haya (Antonia Pascual); Hospital General de Alicante (Fabián Tarín, María Tasso); Hospital Virgen FUNDING de la Arrixaca (María Victoria Martinez-Sánchez, José Antonio Campillo, Eduardo Ramos-Eibal, Esther Llinares, Ana María This study has been supported by a grant from Instituto de Galera, María del Mar Bermúdez); Hospital Vall d’Hebrón (Anna Salud Carlos III (PI12/02417; PI16/00246), integrated into Plan Varela-Magallón); Hospital Virgen de las Nieves (Francisco Ruiz- Nacional de I+D+I and co-funded by ISCIII—Subdirección Cabello, Pilar Jiménez, Ma José Ortega); Hospital Reina Sofía General de Evaluación y Fomento de la Investigación Sanitaria— (Carmen Martínez); Hospital Virgen del Rocío (José Antonio and Fondo Europeo de Desarrollo Regional (FEDER), CIBERER. Pérez-Simón); Hospital Gregorio Marañón (Carolina Martínez- Laperche); Hospital Santa Creu i Sant Pau (Josep Nomdedéu); Hospital Virgen de la Salud (Nerea Domínguez); Hospital ACKNOWLEDGMENTS Maternoinfantil de Badajoz (María Dolores de la Maya, María We are indebted to patients and families and Obra Social Belén Moreno); Hospital Marqués de Valdecillas (Mónica López- from Hospital Sant Joan de Déu, Força Miquel, Candela Duarte), Complejo Hospitalario de Navarra (María Sagaseta de polsera solidària, Pirata Barballum, Magic, and Mua projects Ilurdoz); Hospital Virgen de la Macarena (David García, Helga for their support. We are also indebted with Fundación Cris Benítez, Ana Fernández-Teijeiro); Hospital Son Espases (Marta contra el cáncer, Fundación Unoentrecienmil and Asociación Bernués, Laia Ferrés, José Antonio Salinas). Pablo Ugarte. We also want to thank the excellent technical support from Ms Camino Estella and Ms Lidia Ruiz. We SUPPLEMENTARY MATERIAL are indebted to the Biobanc de l’Hospital Infantil Sant Joan de Déu per a la Investigació, integrated into the The Supplementary Material for this article can be found Spanish Biobank Network of ISCIII for the sample and online at: https://www.frontiersin.org/articles/10.3389/fped. data procurement. 2020.614521/full#supplementary-material REFERENCES 7. Bruggemann M, Kotrova M. Minimal residual disease in adult ALL: technical aspects and implications for correct clinical interpretation. Blood Adv. (2017) 1. Teachey DT, Pui CH. Comparative features and outcomes between paediatric 1:2456–66. doi: 10.1182/bloodadvances.2017009845 T-cell and B-cell acute lymphoblastic leukaemia. Lancet Oncol. (2019) 8. Brüggemann M, Kotrová M, Knecht H, Bartram J, Boudjogrha M, Bystry V, 20:e142–54. doi: 10.1016/S1470-2045(19)30031-2 et al. Standardized next-generation sequencing of immunoglobulin and T- 2. Schrappe M, Hunger SP, Pui CH, Saha V, Gaynon PS, Baruchel A, cell receptor gene recombinations for MRD marker identification in acute et al. Outcomes after Induction Failure in Childhood Acute Lymphoblastic lymphoblastic leukaemia; a EuroClonality-NGS validation study. Leukemia. Leukemia. N Engl J Med. (2012) 366:1371–81. doi: 10.1056/NEJMoa1110169 (2019) 33:2241–53. doi: 10.1038/s41375-019-0496-7 3. Petit A, Trinquand A, Chevret S, Ballerini P, Cayuela JM, Grardel N, et al. 9. Wood B, Wu D, Crossley B, Dai Y, Williamson D, Gawad C, et al. Oncogenetic mutations combined with MRD improve outcome prediction Measurable residual disease detection by high-throughput sequencing in pediatric T-cell acute lymphoblastic leukemia. Blood. (2018) 131:289– improves risk stratification for pediatric B-ALL. Blood. (2018) 131:1350– 300. doi: 10.1182/blood-2017-04-778829 9. doi: 10.1182/blood-2017-09-806521 4. Hefazi M, Litzow MR. Recent advances in the biology and treatment of T 10. Attarbaschi A, Mann G, Panzer-Grümayer R, Röttgers S, Steiner M, König cell acute lymphoblastic leukemia. Curr Hematol Malig Rep. (2018) 13:265– M, et al. Minimal residual disease values discriminate between low and high 74. doi: 10.1007/s11899-018-0455-9 relapse risk in children with B-cell precursor acute lymphoblastic leukemia 5. Rives S, Estella J, Camós M, García-Miguel P, Verdeguer A, Couselo JM, and an intrachromosomal amplification of chromosome 21: the Austrian and et al. [T-cell pediatric acute lymphoblastic leukemia: analysis of survival and German acute lymphoblastic leukemia berl. J Clin Oncol. (2008) 26:3046– prognostic factors in 4 consecutive protocols of the Spanish cooperative 50. doi: 10.1200/JCO.2008.16.1117 study group SHOP]. Med Clin. (2012) 139:141–9. doi: 10.1016/j.medcli.201 11. Modvig S, Madsen HO, Siitonen SM, Rosthøj S, Tierens A, Juvonen V, et al. 1.12.019 Minimal residual disease quantification by flow cytometry provides reliable 6. Schrappe M, Valsecchi MG, Bartram CR, Schrauder A, Panzer-Grümayer risk stratification in T-cell acute lymphoblastic leukemia. Leukemia. (2019) R, Möricke A, et al. Late MRD response determines relapse risk overall 33:1324–36. doi: 10.1038/s41375-018-0307-6 and in subsets of childhood T-cell ALL: results of the AIEOP-BFM- 12. Tembhare PR, Narula G, Khanka T, Ghogale S, Chatterjee G, Patkar NV, et al. ALL 2000 study. Blood. (2011) 118:2077–84. doi: 10.1182/blood-2011-03- Post-induction measurable residual disease using multicolor flow cytometry is 338707 strongly predictive of inferior clinical outcome in the real-life management of Frontiers in Pediatrics | www.frontiersin.org 10 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL childhood T-cell acute lymphoblastic leukemia: a study of 256 patients. Front 29. Jenkinson S, Goulden N, Vora A, Linch DC, Gale RE. Impact of Oncol. (2020) 10:577. doi: 10.3389/fonc.2020.00577 PTEN abnormalities on outcome in pediatric patients with T-cell acute 13. Girardi T, Vicente C, Cools J, De Keersmaecker K. The genetics lymphoblastic leukemia treated on the MRC UKALL2003 trial. Leukemia. and molecular biology of T-ALL. Blood. (2017) 129:1113– (2016) 30:39–47. doi: 10.1038/leu.2015.206 23. doi: 10.1182/blood-2016-10-706465 30. Ribera J, Zamora L, Morgades M, Mallo M, Solanes N, Batlle M, 14. Gianni F, Belver L, Ferrando A. The genetics and mechanisms of T- et al. Copy number profiling of adult relapsed B-cell precursor acute cell acute lymphoblastic leukemia. Cold Spring Harb Perspect Med. (2019) lymphoblastic leukemia reveals potential leukemia progression mechanisms. 10:a035246. doi: 10.1101/cshperspect.a035246 Genes Chromosom Cancer. (2017) 56:810–20. doi: 10.1002/gcc.22486 15. Liu Y, Easton J, Shao Y, Maciaszek J, Wang Z, Wilkinson MR, et al. The 31. Genescà E, Lazarenkov A, Morgades M, Berbis G, Ruíz-Xivillé N, Gómez- genomic landscape of pediatric and young adult T-lineage acute lymphoblastic Marzo P, et al. Frequency and clinical impact of CDKN2A/ARF/CDKN2B leukemia. Nat Genet. (2017) 49:1211–18. doi: 10.1038/ng.3909 gene deletions as assessed by in-depth genetic analyses in adult 16. Belver L, Ferrando A. The genetics and mechanisms of T T cell acute lymphoblastic leukemia. J Hematol Oncol. (2018) cell acute lymphoblastic leukaemia. Nat Rev Cancer. (2016) 11:9–12. doi: 10.1186/s13045-018-0639-8 16:494–507. doi: 10.1038/nrc.2016.63 32. Van Vlierberghe P, Ambesi-Impiombato A, De Keersmaecker K, Hadler M, 17. Kimura S, Seki M, Yoshida K, Shiraishi Y, Akiyama M, Koh K, Paietta E, Tallman MS, et al. Prognostic relevance of integrated genetic et al. NOTCH1 pathway activating mutations and clonal evolution in profiling in adult T-cell acute lymphoblastic leukemia. Blood. (2013) 122:74– pediatric T-cell acute lymphoblastic leukemia. Cancer Sci. (2019) 110:784– 82. doi: 10.1182/blood-2013-03-491092 94. doi: 10.1111/cas.13859 33. Marks DI, Paietta EM, Moorman AV, Richards SM, Buck G, DeWald 18. Kox C, Zimmermann M, Stanulla M, Leible S, Schrappe M, Ludwig W- G, et al. T-cell acute lymphoblastic leukemia in adults: clinical features, D, et al. The favorable effect of activating NOTCH1 receptor mutations immunophenotype, cytogenetics, and outcome from the large randomized on long-term outcome in T-ALL patients treated on the ALL-BFM 2000 prospective trial (UKALL XII/ECOG 2993). Blood. (2009) 114:5136– protocol can be separated from FBXW7 loss of function. Leukemia. (2010) 45. doi: 10.1182/blood-2009-08-231217 24:2005–13. doi: 10.1038/leu.2010.203 34. Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A, et al. 19. Asnafi V, Buzyn A, Le Noir S, Baleydier F, Simon A, Beldjord K, et al. Proposals for the immunological classification of acute leukemias. European NOTCH1/FBXW7 mutation identifies a large subgroup with favorable group for the immunological characterization of leukemias (EGIL). Leukemia. outcome in adult T-cell acute lymphoblastic leukemia (T-ALL): a group for (1995) 9:1783–6. research on adult acute lymphoblastic leukemia (GRAALL) study. Blood. 35. van Dongen JJM, Orfao A. EuroFlow: resetting leukemia and lymphoma (2009) 113:3918–24. doi: 10.1182/blood-2008-10-184069 immunophenotyping. Basis for companion diagnostics and personalized 20. Jenkinson S, Koo K, Mansour MR, Goulden N, Vora A, Mitchell C, et al. medicine. Leukemia. (2012) 26:1899–907. doi: 10.1038/leu.2012.121 Impact of NOTCH1/FBXW7 mutations on outcome in pediatric T-cell acute 36. Weng AP, Ferrando AA, Lee W, Morris JP, Silverman LB, Sanchez-Irizarry C, lymphoblastic leukemia patients treated on the MRC UKALL 2003 trial. et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic Leukemia. (2013) 27:41–7. doi: 10.1038/leu.2012.176 leukemia. Science. (2004) 306:269–71. doi: 10.1126/science.1102160 21. Zuurbier L, Homminga I, Calvert V, te Winkel ML, Buijs-Gladdines JGC 37. R Core Team. R: A Language and Environment for Statistical Computing. a M, Kooi C, et al. NOTCH1 and/or FBXW7 mutations predict for initial (2019). Available online at: https://www.r-project.org/ good prednisone response but not for improved outcome in pediatric T-cell 38. Teachey DT, O’Connor D. How I treat newly diagnosed T-cell acute acute lymphoblastic leukemia patients treated on DCOG or COALL protocols. lymphoblastic leukemia and T-cell lymphoblastic lymphoma in children. Leukemia. (2010) 24:2014–22. doi: 10.1038/leu.2010.204 Blood. (2020) 135:159–66. doi: 10.1182/blood.2019001557 22. Clappier E, Collette S, Grardel N, Girard S, Suarez L, Brunie G, et al. 39. Möricke A, Reiter A, Zimmermann M, Gadner H, Stanulla M, Dördelmann NOTCH1 and FBXW7 mutations have a favorable impact on early response M, et al. Risk-adjusted therapy of acute lymphoblastic leukemia can decrease to treatment, but not on outcome, in children with T-cell acute lymphoblastic treatment burden and improve survival: treatment results of 2169 unselected leukemia (T-ALL) treated on EORTC trials 58881 and 58951. Leukemia. pediatric and adolescent patients enrolled in the trial ALL-BFM 95. Blood. (2010) 24:2023–31. doi: 10.1038/leu.2010.205 (2008) 111:4477–89. doi: 10.1182/blood-2007-09-112920 23. Yuan L, Lu L, Yang Y, Sun H, Chen X, Huang Y, et al. Genetic mutational 40. Aricò M, Valsecchi MG, Conter V, Rizzari C, Pession A, Messina C, et al. profiling analysis of T cell acute lymphoblastic leukemia reveal mutant Improved outcome in high-risk childhood acute lymphoblastic leukemia FBXW7 as a prognostic indicator for inferior survival. Ann Hematol. (2015) defined by prednisone-poor response treated with double berlin-frankfurt- 94:1817–28. doi: 10.1007/s00277-015-2474-0 muenster protocol ii. Blood. (2002) 100:420–6. doi: 10.1182/blood.V100.2.420 24. Zuurbier L, Petricoin EF, Vuerhard MJ, Calvert V, Kooi C, Buijs- 41. Barba P, Morgades M, Montesinos P, Gil C, Fox M, Ciudad J, et al. Gladdines JGC, et al. The significance of PTEN and AKT aberrations Increased survival due to lower toxicity for high-risk T-cell acute in pediatric T-cell acute lymphoblastic leukemia. Haematologica. (2012) lymphoblastic leukemia patients in two consecutive pediatric-inspired 97:1405–13. doi: 10.3324/haematol.2011.059030 PETHEMA trials. Eur J Haematol. (2019) 102:79–86. doi: 10.1111/ej 25. Bandapalli OR, Zimmermann M, Kox C, Stanulla M, Schrappe M, h.13178 Ludwig WD, et al. NOTCH1 activation clinically antagonizes the 42. Liu RB, Guo JG, Liu TZ, Guo CC, Fan XX, Zhang X, et al. Meta-analysis unfavorable effect of PTEN inactivation in BFM-treated children with of the clinical characteristics and prognostic relevance of NOTCH1 and precursor T-cell acute lymphoblastic leukemia. Haematologica. (2013) FBXW7 mutation in T-cell acute lymphoblastic leukemia. Oncotarget. (2017) 98:928–36. doi: 10.3324/haematol.2012.073585 8:66360–70. doi: 10.18632/oncotarget.18576 26. Kindler T, Cornejo MG, Scholl C, Liu J, Leeman DS, Haydu JE, et al. K- 43. Noronha EP, Marques LVC, Andrade FG, Thuler LCS, Terra-Granado E, RasG12D-induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 Pombo-de-Oliveira MS. The profile of immunophenotype and genotype mutations and are sensitive to gamma-secretase inhibitors. Blood. (2008) aberrations in subsets of pediatric T-cell acute lymphoblastic leukemia. Front 112:3373–82. doi: 10.1182/blood-2008-03-147587 Oncol. (2019) 9:316. doi: 10.3389/fonc.2019.00316 27. Perentesis JP, Bhatia S, Boyle E, Shao Y, Shu XO, Steinbuch 44. Erbilgin Y, Sayitoglu M, Hatirnaz O, Dogru O, Akcay A, Tuysuz G, M, et al. RAS oncogene mutations and outcome of therapy et al. Prognostic significance of NOTCH1 and FBXW7 mutations in for childhood acute lymphoblastic leukemia. Leukemia. (2004) pediatric T-ALL. Dis Markers. (2010) 28:353–60. doi: 10.1155/2010/7 18:685–92. doi: 10.1038/sj.leu.2403272 40140 28. Trinquand A, Tanguy-Schmidt A, Abdelali RB, Lambert J, Beldjord K, 45. Park MJ, Taki T, Oda M, Watanabe T, Yumura-Yagi K, Kobayashi R, Lengliné E, et al. Toward a NOTCH1/FBXW7/RAS/PTEN-based oncogenetic et al. FBXW7 and NOTCH1 mutations in childhood T cell acute risk classification of adult T-cell acute lymphoblastic leukemia: a group for lymphoblastic leukaemia and T cell non-Hodgkin lymphoma. Br research in adult acute lymphoblastic leukemia study. J Clin Oncol. (2013) J Haematol. (2009) 145:198–206. doi: 10.1111/j.1365-2141.2009.0 31:4333–42. doi: 10.1200/JCO.2012.48.5292 7607.x Frontiers in Pediatrics | www.frontiersin.org 11 February 2021 | Volume 8 | Article 614521 Vega-García et al. Prognostic Risk-Factors in Pediatric T-ALL 46. Bonn BR, Rohde M, Zimmermann M, Krieger D, Oschlies I, speaker fees, and travel and accommodation support from Celgene (outside Niggli F, et al. Incidence and prognostic relevance of genetic the submitted work); advisory board honorarium, speaker fees, and travel and variations in T-cell lymphoblastic lymphoma in childhood and accommodation support from Sobi (outside the submitted work). SR reports adolescence. Blood. (2013) 121:3153–60. doi: 10.1182/blood-2012-12-4 advisory board honorarium, speaker fees, and travel and accommodation support 74148 from Jazz Pharma and Shire/Servier; personal fees, advisory board honorarium, 47. Neumann M, Vosberg S, Schlee C, Heesch S, Schwartz S, Gökbuget N, speaker fees, and travel and accommodation support from Novartis (outside the et al. Mutational spectrum of adult T-ALL. Oncotarget. (2015) 6:2754– submitted work); advisory board honorarium, and travel and accommodation 66. doi: 10.18632/oncotarget.2218 support from Amgen (outside the submitted work); advisory board honorarium, 48. Yeh TC, Liang DC, Liu HC, Jaing TH, Chen SH, Hou JY, et al. speaker fees, and travel and accommodation support from Celgene (outside Clinical and biological relevance of genetic alterations in pediatric T-cell the submitted work). MC discloses talk fees from Shire/Servier outside the acute lymphoblastic leukemia in Taiwan. Pediatr Blood Cancer. (2019) presented work. 66:e27496. doi: 10.1002/pbc.27496 The remaining authors declare that the research was conducted in the absence of Conflict of Interest: MT reports travel and accommodation support from any commercial or financial relationships that could be construed as a potential Novartis (outside the submitted work); travel and accommodation support from conflict of interest. Jazz Pharma and Shire/Servier (outside the submitted work); and travel and accommodation support from Amgen (outside the submitted work). JLF is a Copyright © 2021 Vega-García, Perez-Jaume, Esperanza-Cebollada, Vicente- consultant/advisory member for Amgen, Jazz Pharmaceuticals, and Novartis Garcés, Torrebadell, Jiménez-Velasco, Ortega, Llop, Abad, Vagace, Minguela, (outside the submitted work), receives honoraria for speaking at symposia from Pratcorona, Sánchez-Garcia, García-Calderón, Gómez-Casares, Martín-Clavero, Amgen, Servier, Jazz Pharmaceuticals, and Pfizer (outside the submitted work) and Escudero, Riñón Martinez-Gallo, Muñoz, Velasco, García-Morin, Català, Pascual, support for attending symposia from Servier and Jazz Pharmaceuticals (outside Velasco, Fernández, Lassaletta, Fuster, Badell, Molinos-Quintana, Molinés, Guerra- the submitted work). AM discloses talk fees from Jazz Pharma and Shire/Servier García, Pérez-Martínez, García-Abós, Robles Ortiz, Pisa, Adán, Díaz de Heredia, outside the presented work and reports travel and accommodation support from Dapena, Rives, Ramírez-Orellana and Camós. This is an open-access article Jazz Pharma, Shire/Servier, and Novartis (outside the submitted work). JD reports distributed under the terms of the Creative Commons Attribution License (CC BY). advisory board honorarium, speaker fees, and travel and accommodation support The use, distribution or reproduction in other forums is permitted, provided the from Jazz Pharma and Shire/Servier; personal fees, advisory board honorarium, original author(s) and the copyright owner(s) are credited and that the original speaker fees, and travel and accommodation support from Novartis (outside the publication in this journal is cited, in accordance with accepted academic practice. submitted work); advisory board honorarium, and travel and accommodation No use, distribution or reproduction is permitted which does not comply with these support from Amgen (outside the submitted work); advisory board honorarium, terms. Frontiers in Pediatrics | www.frontiersin.org 12 February 2021 | Volume 8 | Article 614521