BR Biomedical Reports 2049-9434 2049-9442 D.A. Spandidos BR-0-0-01353 10.3892/br.2020.1353 Articles Panel-based next-generation sequencing facilitates the characterization of childhood acute myeloid leukemia in clinical settings IshidaHisashi 1 IguchiAkihiro 2 AoeMichinori 3 NishiuchiRitsuo 4 MatsubaraTakehiro 5 KeinoDai 6 SanadaMasashi 7 ShimadaAkira 1 Department of Pediatrics/Pediatric Hematology and Oncology, Okayama University Hospital, Okayama 700-8558, Japan Department of Pediatrics, Hokkaido University Hospital, Sapporo, Hokkaido 060-8648, Japan Division of Medical Support, Okayama University Hospital, Okayama 700-8558, Japan Department of Pediatrics, Kochi Health Sciences Center, Kochi 781-8555, Japan Division of Biobank, Center for Comprehensive Genomic Medicine, Okayama University Hospital, Okayama 700-8558, Japan Department of Pediatrics, St. Marianna University School of Medicine Hospital, Kawasaki, Kanagawa 216-8511, Japan Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Aichi 460-0001, Japan Correspondence to: Professor Akira Shimada, Department of Pediatrics/Pediatric Hematology and Oncology, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan pajj236e@okayama-u.ac.jp 11 2020 28 08 2020 13 5 46 11 01 2020 10 07 2020 Copyright: © Ishida et al. 2020 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Acute myeloid leukemia (AML) accounts for ~20% of pediatric leukemia cases. The prognosis of pediatric AML has been improved in recent decades, but it trails that of most other types of pediatric cancer, with mortality rates of 30-40%. Consequently, newer more targeted drugs are required for incorporation into treatment plans. These newer drugs selectively target AML cells with specific gene alterations. However, there are significant differences in genetic alterations between adult and pediatric patients with AML. In the present study, inexpensive and rapid next-generation sequencing (NGS) of >150 cancer-related genes was performed for matched diagnostic, remission and relapse (if any) samples from 27 pediatric patients with AML. In this analysis, seven genes were recurrently mutated. KRAS was mutated in seven patients, NRAS was mutated in three patients, and KIT, GATA1, WT1, PTPN11, JAK3 and FLT3 were each mutated in two patients. Among patients with relapsed AML, six harbored KRAS mutations at diagnosis; however, four of these patients lost these mutations at relapse. Additionally, two genetic alterations (FLT3-ITD and TP53 alterations) were detected among patients who eventually relapsed, and these mutations are reported to be adverse prognostic factors for adult patients with AML. This panel-based, targeted sequencing approach may be useful in determining the genetic background of pediatric AML and improving the prediction of treatment response and detection of potentially targetable gene alterations. RAS pathway mutations were highly unstable at relapse; therefore, these mutations should be chosen as a target with caution. Incorporating this panel-based NGS approach into the clinical setting may allow for a patient-oriented strategy of precision treatment for childhood AML.

 leukemia pediatric acute myeloid leukemia molecular genetics precision medicine Introduction

Acute myeloid leukemia (AML) accounts for ~20% of all cases of pediatric leukemia (1). Although the prognosis of pediatric AML has improved in recent decades, it trails that of other types of pediatric cancer, as 30-40% of children with AML eventually succumb to the disease (1-3). The treatment strategies for pediatric AML include intensive multimodal chemotherapy with or without stem cell transplantation, and cytarabine and anthracyclines have remained the primary choices of chemotherapy for >30 years (1,2,4,5). As clinical outcomes have not improved, even with intensive contemporary chemotherapeutic regimens and/or stem cell transplantation, newer targeted drugs are required for incorporation into treatment plans (5,6).

Recently, various newer targeted therapies have emerged, most of which target AML cells with specific genetic alterations (7). Among them, FLT3 inhibitors, such as midostaurin and gilteritinib, and IDH inhibitors, such as enasidenib and ivosidenib have already been approved for use in clinical settings (7). These newer drugs selectively target AML cells with specific features; hence, the genomic characterization of AML cells is becoming increasingly important in the clinical setting. Using next-generation sequencing (NGS), several studies have reported on the value of performing NGS for adult patients with AML (8-12). However, there are comparatively fewer reports focusing on childhood AML, particularly in cases of relapsed AML (13,14).

In the present study, panel-based, targeted NGS for the molecular characterization of AML cells from pediatric patients was retrospectively performed. The objective of this study was to determine whether it was possible to obtain clinically useful information for children with AML through this NGS approach.

 Materials and methods <sec> <title>Patients

A total of 27 children aged 0-18 years who were diagnosed with AML between January 2000 and December 2017 in Okayama University Hospital, Kochi Health Sciences Center, St. Marianna University School of Medicine Hospital or Hokkaido University Hospital were enrolled in the present study. The ratio of boys to girls was 17:10 in the present cohort and the median age at diagnosis was 6 years (range, 0 months to 15 years). The treatment protocols were diverse, including those from the Japanese Pediatric Leukemia/Lymphoma Study Group AML-99(15), AML-05(16) and AML-12 studies, and are listed in Table I. The standard chimeric fusion gene screening varied has changed over time in Japan. AML-05 protocol included the PCR-based detection of eight frequent chimeric gene fusions; RUNX1-RUNXT1, CBFB-MYH11, KMT2A-MLLT3, KMT2A-MLLT4, KMT2A-MLLT1, FUS-ERG, NUP98-HOXA9 and PML-RARA, and the AML-12 protocol included eight gene fusions; RUNX1-RUNXT1, CBFB-MYH11, KMT2A-MLLT3, KMT2A-MLLT4, BCR-ABL, FUS-ERG, NUP98-NSD1 and PML-RARA. In our previous study, the clinical courses of UPN 6 and UPN 25 was reported (17,18). The present study was performed in accordance with the Declaration of Helsinki and other applicable guidelines (19-21). The present study was approved by the Institutional Ethics Committee of Okayama University Hospital, and informed consent was obtained the patients and/or their legal guardians.

 DNA isolation

Somatic DNA was obtained from bone marrow samples at diagnosis and each episode of relapse, whereas germline DNA was obtained from a buccal swab or peripheral blood in CR status. DNA was extracted using a QIAamp DNA Blood Mini kit (Qiagen, Inc.) and quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.) and a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) according to the manufacturers' protocol.

 Targeted NGS approach

Targeted sequencing of >150 cancer-related genes was performed as described previously (22-24). The targeted gene lists are shown in Table SI and patient allocation is shown in Table SII. These gene panels were generated using an online design tool for HaloPlex (Agilent Technologies, Inc.), and target enrichment was performed using the HaloPlex standard protocol. Samples were then sequenced using MiSeq (Illumina, Inc.). Read alignment to the hg19 reference genome was performed using Burrows-Wheeler Aligner (bio-bwa.sourceforge.net) and variant calling was performed using SureCall version 3.0 (Agilent Technologies, Inc.).

 Variant prioritization and assessment of pathogenicity

Synonymous or non-coding variants and single nucleotide polymorphisms reported with a frequency of >1% in various databases (dbSNP, 1000gp and Human Genetic Variation Database) were excluded. Variant bases that had >5 reads in each sample were used for the next step. Genetic variations that were constantly detected from diagnostic, remission and relapse samples with a variant allele frequency (VAF) ≥0.2 were regarded as candidate germline alterations. Genetic alterations that were rarely detected, or not detected at all, from remission samples but were detected from diagnostic and/or relapse samples with a VAF ≥0.05 were regarded as candidate somatic alterations with reference to a previous study (25). To exclude the possibility of false-positive findings, differences in VAF between normal and diagnostic/relapse samples were assessed using a Fisher's exact test. P<0.01 was considered to indicate a statistically significant difference. Finally, for germline and somatic alterations, the read quality was checked using IGV software version 2.3 (Broad Institute).

 Results <sec> <title>Patient characteristics

The clinical information of the analyzed patients is shown in Table I. All patients were Japanese. The present cohort included one patient with Down-syndrome (UPN 8) and no other patients had a known underlying congenital condition. Remission samples were obtained by buccal smear from patient No. 2 and the sample collection was performed during remission. All relapsed patients lost their first remission within 24 months; 6 out of 15 patients experienced their relapse within 12 months, which is considered to be an adverse prognostic factor for survival (26).

 Descriptive results from sequencing runs

The average number of total reads was 1,917,277 (range, 998,341-4,333,332) and the average read length was 116-136. The read depth in analyzable target regions per sample ranged from 168-757x. In total, 70.49-97.51% of analyzable regions were covered by at least 20 reads, 66.72-95.24% were covered by at least 50 reads and 58.15-91.05% were covered by at least 100 reads. These quality metrics data were obtained from analysis using SureCall version 3.0 software (Agilent Technologies, Inc.) and the details are shown in Table SII.

The results of NGS are shown in Table II. A total of 26 single nucleotide variations (SNVs) and insertions/deletions (indels) were identified at diagnosis, and 22 SNVs and indels at relapse for 15 patients with relapsed AML, as well as 12 SNVs and indels for the leukemia samples of 12 patients without relapse.

 Somatic genetic alterations at diagnosis and AML subtypes

In the present study, seven genes were recurrently mutated. KRAS was mutated in 7 patients, NRAS was mutated in 3 patients, and KIT, GATA1, WT1, PTPN11, JAK3 and FLT3 were each mutated in 2 patients. As previously reported, KIT was mutated in patients with core-binding factor AML (UPN 19 and UPN 27), and GATA1 was mutated in patients with acute megakaryoblastic leukemia with or without Down-syndrome (UPN 5 and UPN 8) (27-29). The present cohort included 2 patients with mixed-phenotype acute leukemia, and these patients harbored mutations previously reported in a larger study (30). Other mutations were not apparently associated with a specific type of AML. Most detected mutations have been reported in the Catalogue Of Somatic Mutations In Cancer database (cancer.sanger.ac.uk/cosmic), but some alterations were not. These mutations were thought to be variants of unknown significance and are indicated with a superscripted letter in Table II.

 Prognostic genetic alterations detected at diagnosis

According to the European LeukemiaNet (ELN) guideline, several factors are associated with the prognosis of AML (31). Among these, two high-risk genetic alterations (FLT3-ITD in patient 6 and TP53 alteration in patient 15) and one low-risk genetic alteration (CEBPA mutation in patient 11) were added to the known cytogenetic risk factors (Table III). According to the guidelines, RUNX1 or ASXL1 alterations are indicated not to be used as adverse prognostic markers if they are present concurrently with favorable-risk AML subtypes. However, TP53 alterations are regarded as an independent adverse prognostic factor, thus UPN 15 was placed in the ELN high-risk group. The gene alterations which were defined in the ELN guideline or recurrently detected in the present study are summarized in Fig. 1.

 Mutational changes between diagnosis and relapse

A total of 15 patients who experienced relapse were analyzed. Among these, six harbored KRAS mutations at diagnosis. However, four of the six patients lost these mutations at relapse. None of the patients gained new RAS pathway mutations at relapse.

UPN 11 had a CEBPA p.Q312HR insertion-type alteration at diagnosis, and this alteration appeared to be homozygous as its VAF was notably high (0.99). At relapse however, the VAF of CEBPA p.Q312HR alteration decreased, and a new p.D53fs alteration appeared, leading to a ‘double hit’ status.

 Germline variations

In the present study, matched samples at diagnosis, remission and relapse (if any) among patients with childhood AML was analyzed, and this approach enabled detection of germline variations. Although candidates of germline variations were detected in 8 patients, none were regarded as pathogenic or likely pathogenic according to published recommendations (19,21). These candidate genes are listed in Table SIII.

 Discussion

In the present study, matched samples obtained from pediatric patients with AML at diagnosis, remission and relapse (if any) were analyzed using a panel-based NGS method. Several studies have reported the utility of NGS for analysis of AML in adult patients (8-12). However, there are comparatively fewer reports focusing on childhood AML, particularly in cases of relapsed AML (13,14).

The utility of NGS should be discussed separately in adult and pediatric patients. As was shown in the present study, there are significant differences in genetic alterations between adult and pediatric patients with AML. Whereas mutations in epigenetic components or spliceosome complexes are common among adults with AML (11), these mutations were notably less common amongst children with AML, based on the results of the present study. However, large structural aberrations such as chromosomal translocations are more common amongst children with AML (11,14). As a result, disease stratification guidelines or newer drugs developed for adults with AML may not always be suitable for children.

The panel-based NGS strategy has been used several times in our previous studies for hematological malignancies, and the results of this method have been compared with conventional approaches, such as the multiplex ligation-dependent probe amplification or Sanger sequencing in our previous studies (22-24). Based on the panel-based NGS approach, several prognostic genetic alterations for patients with AML were detected in the present study. The ELN guidelines identified several cytogenetic alterations and a smaller number of genetic mutations as prognostic factors. In the present cohort, additional cytogenetic risk factors possessed prognostic implications than the genetic mutations. Using this approach, two high-risk genetic mutations were detected amongst patients who relapsed but did not possess any cytogenetic risk factors (UPN 6 and UPN 15). Conversely, none of the non-relapsed patients possessed adverse prognostic genetic mutations, and non-relapsed patients were enriched in low risk genetic alternations defined in the ELN guidelines. Thus, it may be possible to more accurately predict the prognosis of patients with AML using this approach. However, as noted above, there are significant differences in genetic alterations between adult and pediatric patients with AML. Previous larger studies suggested that current guidelines, including the ELN guidelines, are not adequate for children with AML (14,32,33), thus, there is a need for the development of a pediatric-specific guidelines for more precise stratification.

FLT3-ITD has a prognostic impact in pediatric AML (34,35), and this alteration was detected using the panel-based NGS method. However, the clinical impact of FLT3-ITD has been reported to be modulated by other sequence aberrations (WT1 mutation, NUP98-NSD1 or NPMI mutations for adult AML) (14,36). In this context, panel-based sequencing may be more useful than conventional approaches that detect only FLT3-ITD. However, the ELN guidelines recommend the use of DNA fragment analysis to determine the ratio of FLT3-ITD and prognosis. To confirm the usefulness of the NGS approach, a direct comparison of standard procedures and the NGS approach is required.

The number of genes that should be assessed has increased; however, one large study found that a limited number of genes are recurrently mutated in pediatric patients with AML (14); where several genetic analytical methods, including whole-genome and targeted DNA sequencing were performed. Mutations in only 5 genes (FLT3, NPM1, WT1, CEBPA and KIT) were present in >5% of patients, and <40 gene mutations were reported in >2% of patients. This previous study illustrated the need to focus on these 40 genes to detect recurrent gene mutations in AML cells obtained from pediatric patients, and that panel-based sequencing is an ideal approach in a clinical setting. Furthermore, Morita et al (37) reported that the clearance of somatic mutation at remission was associated with significantly improved survival and a lower risk of relapse. This strategy requires a sufficient read depth to detect mutations with a VAF <1%; contrarily, the approach used in the present study could not reach that read depth due to low throughput and relatively high number of targeted genes. Limiting the number of targeted genes to 40 recurrently mutated genes will increase the read depth and potentially enable detection of gene alterations with lower VAFs.

The panel-based approach used in the present study also offers potentially useful information regarding targetable genetic alterations. Cytotoxic chemotherapy primarily based on cytarabine and anthracyclines with or without stem cell transplantation has long been the mainstay of AML treatment (1). The curative rate of pediatric AML steadily improves with increasing doses of these drugs; however, this leads to substantial treatment-related complications in vulnerable pediatric populations (1,16,26). Hence, newer targeted therapies are desired. These targeted drugs include midostaurin, gilteritinib, enasidenib and ivosidenib, which target leukemic cells with specific genetic alterations such as FLT3, KIT, MEK, DOT1L or BET alterations (5,6); hence, genomic characterization of AML cells is becoming increasingly important in the clinical setting. Among these potentially targetable gene alterations, 6 patients had RAS pathway mutations at the time of AML diagnosis. However, in the present cohort, a notably high percentage of patients (four out of six cases, 66.7%) lost the KRAS mutations at relapse. Thus, RAS pathway mutations should be chosen with caution as treatment targets.

The present study has several limitations. First, the study was retrospective, and it was not possible to clarify whether dose increases in patients who had high-risk features would improve their prognoses. Second, the method used in the present study has a disadvantage of comparatively low throughput. Third, potential false positives were excluded, thus several relevant genetic alterations may have been missed. To detect minor clones with low variant frequencies at diagnosis, the number of genes to be targeted should be limited, as noted above. The genetic events including TP53 loss of heterozygosity in UPN 15 should have also been illustrated using other experiments, such as fluorescent in situ hybridization, but this could not be achieved due to the sample availability and quality. Relatively small patient numbers is another disadvantage of the present study. Furthermore, the ELN guidelines were established for adult patients; hence its validity in a larger cohort of pediatric patients with AML requires validation.

In summary, the panel-based targeted sequencing approach used in the present study may be useful for revealing the genetic background of pediatric AML, and may facilitate the precise prediction of patient prognosis and detection of druggable gene alterations. Incorporating this method into the clinical setting may enable a patient-oriented precision strategy for treatment of childhood AML.

 Supplementary Material HaloPlex custom panels used in the present study. Quality metrics of next-generation sequencing. Candidate germline variants detected in the present study. Acknowledgements

Not applicable.

 Funding

This present study was supported by grants from MEXT/JSPS KAKENHI (grant no. JP 20K08157).

 Availability of data and materials

The datasets generated and/or analyzed during the present study could not be submitted to a public curated database as the informed consent obtained does not include unrestricted disclosure of sequencing data. Instead, these data are available from the corresponding author upon reasonable request.

 Authors' contributions

HI and AS wrote the manuscript. HI, MA, TM, MS and AS performed the genetic analysis and interpreted the results. HI, AI, RN, DK and AS performed patient care and collected the clinical data. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

The present study was approved by The Institutional Review Board of Okayama University Hospital (Okayama, Japan). Informed consent was obtained from the patients and/or their legal guardians.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References TagaTTomizawaDTakahashiHAdachiSAcute myeloid leukemia in children: Current status and future directionsPediatr Int58718020162664570610.1111/ped.12865 RascheMZimmermannMBorschelLBourquinJPDworzakMKlingebielTLehrnbecherTCreutzigUKlusmannJHReinhardtDSuccesses and challenges in the treatment of pediatric acute myeloid leukemia: A retrospective analysis of the AML-BFM trials from 1987 to 2012Leukemia322167217720182955083410.1038/s41375-018-0071-7 PikmanYStegmaierKTargeted therapy for fusion-driven high-risk acute leukemiaBlood1321241124720183004980910.1182/blood-2018-04-784157 DohnerHWeisdorfDJBloomfieldCDAcute myeloid leukemiaN Engl J Med3731136115220152637613710.1056/NEJMra1406184 ZwaanCMKolbEAReinhardtDAbrahamssonJAdachiSAplencRDe BontESDe MoerlooseBDworzakMGibsonBECollaborative efforts driving progress in pediatric acute myeloid leukemiaJ Clin Oncol332949296220152630489510.1200/JCO.2015.62.8289 KolbEAMeshinchiSAcute myeloid leukemia in children and adolescents: Identification of new molecular targets brings promise of new therapiesHematology201550751320152663776610.1182/asheducation-2015.1.507 KayserSLevisMJAdvances in targeted therapy for acute myeloid leukaemiaBr J Haematol18048450020182919301210.1111/bjh.15032 DingLLeyTJLarsonDEMillerCAKoboldtDCWelchJSRitcheyJKYoungMALamprechtTMcLellanMDClonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencingNature48150651020122223702510.1038/nature10738 WongTNRamsinghGYoungALMillerCAToumaWWelchJSLamprechtTLShenDHundalJFultonRSRole of TP53 mutations in the origin and evolution of therapy-related acute myeloid leukaemiaNature51855255520152548715110.1038/nature13968 QuekLFergusonPMetznerMAhmedIKennedyAGarnettCJeffriesSWalterCPiechockiKTimbsAMutational analysis of disease relapse in patients allografted for acute myeloid leukemiaBlood Adv119320420162929693510.1182/bloodadvances.2016000760 PapaemmanuilEGerstungMBullingerLGaidzikVIPaschkaPRobertsNDPotterNEHeuserMTholFBolliNGenomic classification and prognosis in acute myeloid leukemiaN Engl J Med3742209222120162727656110.1056/NEJMoa1516192 GreifPAHartmannLVosbergSStiefSMMattesRHellmannIMetzelerKHHeroldTBamopoulosSAKerbsPEvolution of cytogenetically normal acute myeloid leukemia during therapy and relapse: An exome sequencing study of 50 patientsClin Cancer Res241716172620182933020610.1158/1078-0432.CCR-17-2344 ShibaNYoshidaKShiraishiYOkunoYYamatoGHaraYNagataYChibaKTanakaHTeruiKWhole-exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemiaBr J Haematol17547648920162747091610.1111/bjh.14247 BolouriHFarrarJETricheT JrRiesRELimELAlonzoTAMaYMooreRMungallAJMarraMAThe molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactionsNat Med2410311220182922747610.1038/nm.4439 TsukimotoITawaAHoribeKTabuchiKKigasawaHTsuchidaMYabeHNakayamaHKudoKKobayashiRRisk-stratified therapy and the intensive use of cytarabine improves the outcome in childhood acute myeloid leukemia: The AML99 trial from the Japanese childhood AML cooperative study groupJ Clin Oncol274007401320091962049110.1200/JCO.2008.18.7948 TomizawaDTawaAWatanabeTSaitoAMKudoKTagaTIwamotoSShimadaATeruiKMoritakeHExcess treatment reduction including anthracyclines results in higher incidence of relapse in core binding factor acute myeloid leukemia in childrenLeukemia272413241620132367733510.1038/leu.2013.153 HanadaTKanamitsuKChayamaKMiyamuraTKanazawaYMuraokaMWashioKImadaMKageyamaMTakeuchiAA Long-term survivor after congenital acute myeloid leukemia with t(8 ; 16)(p11 ; p13)Acta Med Okayama70313520162689960710.18926/AMO/54001 IwasakiYNishiuchiRAoeMTakahashiTWatanabeHTokorotaniCKikkawaKhimadaAPositive minimal residual disease of FLT3-ITD before hematopoietic stem cell transplantation resulted in a poor prognosis of an acute myeloid leukemiaActa Med Okayama71798320172823801410.18926/AMO/54829 GreenRCBergJSGrodyWWKaliaSSKorfBRMartinCLMcGuireALNussbaumRLO'DanielJMOrmondKEACMG recommendations for reporting of incidental findings in clinical exome and genome sequencingGenet Med1556557420132378824910.1038/gim.2013.73 RichardsSAzizNBaleSBickDDasSGastier-FosterJGrodyWWHegdeMLyonESpectorEStandards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular PathologyGenet Med1740542320152574186810.1038/gim.2015.30 KaliaSSAdelmanKBaleSJChungWKEngCEvansJPHermanGEHufnagelSBKleinTEKorfBRRecommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): A policy statement of the american college of medical genetics and genomicsGenet Med1924925520172785436010.1038/gim.2016.190 IshidaHKanamitsuKWashioKMuraokaMSakakibaraKMatsubaraTKanzakiHShimadaARelapsed infant MLL-rearranged acute lymphoblastic leukemia with additional genetic alterationsPediatr Blood Cancer632059206020162732281610.1002/pbc.26100 AoeMIshidaHMatsubaraTKarakawaSKawaguchiHFujiwaraKKanamitsuKWashioKOkadaKShibakuraMShimadaASimultaneous detection of ABL1 mutation and IKZF1 deletion in Philadelphia chromosome-positive acute lymphoblastic leukemia using a customized target enrichment system panelInt J Lab Hematol4042743620182957554110.1111/ijlh.12805 IshidaHIguchiAAoeMTakahashiTTamefusaKKanamitsuKFujiwaraKWashioKMatsubaraTTsukaharaHPanel-based next-generation sequencing identifies prognostic and actionable genes in childhood acute lymphoblastic leukemia and is suitable for clinical sequencingAnn Hematol9865766820193044680510.1007/s00277-018-3554-8 SunamiKIchikawaHKuboTKatoMFujiwaraYShimomuraAKoyamaTKakishimaHKitamiMMatsushitaHFeasibility and utility of a panel testing for 114 cancer-associated genes in a clinical setting: A hospital-based studyCancer Sci1101480149020193074273110.1111/cas.13969 NakayamaHTabuchiKTawaATsukimotoITsuchidaMMorimotoAYabeHHoribeKHanadaRImaizumiMOutcome of children with relapsed acute myeloid leukemia following initial therapy under the AML99 protocolInt J Hematol10017117920142496164410.1007/s12185-014-1616-9 DuployezNMarceau-RenautABoisselNPetitABucciMGeffroySLapillonneHRennevilleARaguCFigeacMComprehensive mutational profiling of core binding factor acute myeloid leukemiaBlood1272451245920162698072610.1182/blood-2015-12-688705 FaberZJChenXGedmanALBoggsKChengJMaJRadtkeIChaoJRWalshMPSongGThe genomic landscape of core-binding factor acute myeloid leukemiasNat Genet481551155620162779862510.1038/ng.3709 de RooijJDEBranstetterCMaJLiYWalshMPChengJObulkasimADangJEastonJVerboonLJPediatric non-Down syndrome acute megakaryoblastic leukemia is characterized by distinct genomic subsets with varying outcomesNat Genet4945145620172811273710.1038/ng.3772 AlexanderTBGuZIacobucciIDickersonKChoiJKXuBPayne-TurnerDYoshiharaHLohMLHoranJThe genetic basis and cell of origin of mixed phenotype acute leukaemiaNature56237337920183020939210.1038/s41586-018-0436-0 DöhnerHEsteyEGrimwadeDAmadoriSAppelbaumFRBüchnerTDombretHEbertBLFenauxPLarsonRADiagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panelBlood12942444720172789505810.1182/blood-2016-08-733196 Marceau-RenautADuployezNDucourneauBLabopinMPetitARousseauAGeffroySBucciMCuccuiniWFenneteauOMolecular profiling defines distinct prognostic subgroups in childhood AMLHemasphere2e3120183172375910.1097/HS9.0000000000000031 ShibaNYoshidaKHaraYYamatoGShiraishiYMatsuoHOkunoYChibaKTanakaHKaburagiTTranscriptome analysis offers a comprehensive illustration of the genetic background of pediatric acute myeloid leukemiaBlood Adv33157316920193164832110.1182/bloodadvances.2019000404 MeshinchiSAlonzoTAStirewaltDLZwaanMZimmermanMReinhardtDKaspersGJHeeremaNAGerbingRLangeBJRadichJPClinical implications of FLT3 mutations in pediatric AMLBlood1083654366120061691222810.1182/blood-2006-03-009233 ShimadaATakiTTabuchiKTaketaniTHanadaRTawaATsuchidaMHoribeKTsukimotoIHayashiYTandem duplications of MLL and FLT3 are correlated with poor prognoses in pediatric acute myeloid leukemia: A study of the Japanese childhood AML cooperative study groupPediatr Blood Cancer5026426920081776346410.1002/pbc.21318 IveyAHillsRKSimpsonMAJovanovicJVGilkesAGrechAPatelYBhudiaNFarahHMasonJAssessment of minimal residual disease in Standard-risk AMLN Engl J Med37442243320162678972710.1056/NEJMoa1507471 MoritaKKantarjianHMWangFYanYBueso-RamosCSasakiKIssaGCWangSJorgensenJSongXClearance of somatic mutations at remission and the risk of relapse in acute myeloid leukemiaJ Clin Oncol361788179720182970200110.1200/JCO.2017.77.6757

Gene alterations defined in ELN guidelines or recurrently detected in the present study. Distribution of relevant gene mutations. UPN, unique patient number; ELN, European LeukemiaNet; SCT, stem cell transplantation; CR1, first complete remission.

Clinical information of the analyzed patients.

A, Relapsed AML
UPN Age Sex Disease subtype Chimeric gene Karyotyping Initial therapya SCT at CR1 Duration of CR1, months Karyotype at relapse Outcome Institute
1 12 years M FAB M5 KMT2A-MLLT3 46, XY, add(11)(q23), der(21)t(1;21) (q11;q11.2) [9/20], 46, sl, der(19)t(11;19) (q13;p13) [11/20] AML 05 No 24 47, XY, add(11) (q23), +marl Alive OU
2 4 years M FAB M6 (RAEB) None 46, XY, inv(9) (p11q13) Other Yes 7 46, XY, t(2;5)(q33;q31), add(6)(q21), t(6;12)(q13;p13), inv(9)(p11q13) Alive OU
3 5 months M FAB M7 (non-Down-syndrome) None 49, XY, +4, ?t(4;5)(q21;q15), del(12)(p?), +19, +22 AML 12 No 13 50, XY, +4, ?t(4;5)(q21;q15), +8, del(12)(p?), +19, +22 Alive OU
4 10 years M FAB M5 None 48, XY, +16, +19 AML 05 No 9 46, XY, add(7)(q32), ?t(10;11)(p12;q23) Succumbed to disease OU
5 4 years F FAB M7 (non-Down-syndrome) None 46, XX, add(5) (p13), del(6)(q), add(12)(p11.2) Other No 22 46, XX, add(5)(p13), del(6)(q), add(12)(p11.2) Alive OU
6 12 years F FAB M2 None 46, XX AML 05 Yes 7 NA Succumbed to disease KHSC
7 7 years F FAB M2 None 46, XX AML 99 No 10 46, XX Succumbed to disease HU
8 1 year M FAB M7 (Down-syndrome) None 47, XY, +21 Other No 12 45, XY, -10, der(17) t(17;18)(p11;q11), -18, +21 Dead (CR) HU
9 13 years M FAB M4 NA 46, XY, t(6;11) (q27;q23) AML 99 No 18 46, XY, t(6;11)(q27;q23) Succumbed to disease HU
10 1 year M FAB M5b NA 47, XY, t(9;11) (p22;q23), +mar1 AML 99 No 6 NA Alive HU
11 14 years M FAB M1 None 46, XY, del(9)(q?) AML 05 No 18 46, XY Alive HU
12 11 years F T/Myeloid None 46, XX, t(1;2) (p31;p16) AML 05 No 13 46, XX, t(1;2)(p31;p16) Alive HU
13 5 years M FAB M7 (non-Down-syndrome) None 47, XY, del(9) (q12q34), del(12)(p12), +21 Other Yes 19 47, XY, +8, del(9)(q12q34), t(10;17)(q11.2;q25), del(12)(p12) Succumbed to disease HU
14 5 years F FAB M4 None 46, XX, der(2) t(11;10;2)(q21; q11.2;q37), der(10) add(10)(p11.2) t(11;10;2), der(11) t(11;10;2) AML 05 No 24 46, XX Succumbed to disease HU
15 14 years M T/Myeloid None 47, XY, del(4)(q?), +22, inc AML05 No 4 47,XY, del(4)(q?), 22 Alive SMU
B, Non-relapsed AML
UPN Age Sex Disease subtype Chimeric gene Karyotyping Initial therapya SCT at CR1 Duration of CR1, months Karyotype at relapse Outcome Institute
16 10 years M FAB M3 PML-RARA 46, XY, t(15;17) (q22;q11~21) AML99 No - - Alive OU
17 9 years F FAB M3 PML-RARA 46, XX, t(15;17) (q22;q12) AML 99 No - - Alive OU
18 8 months F FAB M5a KMT2A-MLLT10 46, XX, t(10;11) (p12;q23) AML 12 Yes - - Alive OU
19 13 years M FAB M4Eo CBFB-MYH11 47, XY, +8, inv(16)(p13.1q22) AML 05 No - - Alive OU
20 7 years M FAB M2 RUNX1-RUNXT1 45, X, -Y, t(8;21) (q22;q22) AML 05 No - - Alive OU
21 15 years M AML with myelodysplasia-related changes None 46, XY Other Yes - - Dead (CR) OU
22 6 years F FAB M2 CBFB-MYH11 46, XX, inv(16) (p13.1q22) AML 05 No - - Alive OU
23 5 years F FAB M2 RUNX1-RUNXT1 46, XX, t(8;21) (q22;q22) AML 05 No - - Alive OU
24 3 years M FAB M2 RUNX1-RUNXT1 46, XY, t(1;21;8) (p36;q22;q22), del(9)(q13q22) AML 99 No - - Alive OU
25 0 months F FAB M5 KAT6A-CREBBP t(8;16)(p11;p13) AML 99 No - - Alive OU
26 11 months M FAB M5 KMT2A-MLLT3 46, XY AML 99 No - - Alive OU
27 1 year M FAB M2 RUNX1-RUNXT1 46, XY, t(8;21)(q22;q22), del(9)(q?), add(11)(q23) AML 99 No - - Alive OU

aDetails regarding treatment protocols are described in references (15,16). UPN, unique patient number; AML, acute myeloid leukemia; SCT, stem cell transplantation; CR1, first complete remission; OU, Okayama University Hospital; HU, Hokkaido University Hospital; SMU, St. Marianna University School of Medicine Hospital; KHSC, Kochi Health Sciences Center.

Gene alterations detected at diagnosis and relapse.

A, Relapsed AML
UPN Disease subtype SNVs at diagnosis (VAF) SNVs at relapse (VAF)
1 FAB M5 KRASp.G12V (0.296) None
2 FAB M6 (RAEB) PTPN11p.G60V(0.83) PTPN11 p.V45L (0.10) PTPN11p.G60V(0.44)
3 FAB M7 (non-Down-syndrome) KRAS p.A146T (0.09) None
4 FAB M5 None None
5 FAB M7 (non-Down-syndrome) GATA1p.R270W (0.43) KRAS p.G12A (0.43) IKZF1 p.F154Y (0.38)a IKZF1p.L161H (0.38)a GATA1p.R270W(0.23) KRAS p.G12A (0.19) IKZF1p.F154Y(0.17)a IKZF1p.F161H(0.17)a
6 FAB M2 FLT3-ITD (0.572) FLT3-ITD (0.689)
7 FAB M2 PTPN11 p.I479F (0.40) + c.1473-1481dela PTPN11p.I479F (0.35) + c.1473-1481dela
8 FAB M7 (non-Down-syndrome) GATA1 p.P50fs (0.30) GATA1 p.P50fs (0.05)
9 FAB M4 KRAS p.G12V (0.41) SMARCA4p.R979Q (0.06)a MLH1p.A586S (0.06)a
10 FAB M5b KRAS p.G13D (0.32) RUNX1p.Q390fs(0.2)
11 FAB M1 GATA2 p.R362Q (0.11) CEBPAp.Q312HR (0.99)a CEBPAp.D63fs(0.37)a CEBPAp.Q312HR (0.39)a
12 T/Myeloid JAK3 p.L857P (0.10) NOTCH1p.V1721E (0.33) STAT5Bp.N642H(0.11) NF1 p.L2317H (0.05)a JAK3 p.L857P (0.13) NOTCH1p.V1721E (0.33) IL7R p.L243GTARCV (0.10)a VHL p.L85fs (0.11)
13 FAB M7 (non-Down-syndrome) None None
14 FAB M4 U2AF1 p.R35L (0.53) KRAS p.G12D (0.37) KRAS p.G12D (0.23)
15 T/Myeloid TP53 p.K164E (0.998) PHF6 p.R274fs (0.724) NPM1 p.L287fs (0.579) TP53 p.K164E (0.858) PHF6p.R274fs (0.611) NPM1 p.L287fs (0.53) MPLp.A486V(0.064)a
B, Non-relapsed AML
UPN Disease subtype SNVs at diagnosis (VAF) SNVs at relapse (VAF)
16 FAB M3 None -
17 FAB M3 None -
18 FAB M5a None -
19 FAB M4Eo KIT p.D816Y (0.28) -
    WT1 p.H448Y (0.22)a -
    NRAS p.Q61K (0.08) -
20 FAB M2 JAK3 p.M511I (0.13) -
21 AML with myelodysplasia-related changes None -
22 FAB M2 NRAS p.G13D (0.37) -
    WT1 p.D447N (0.34) -
23 FAB M2 NRAS p.Q61K (0.25) -
24 FAB M2 KRAS p.G12D (0.52) -
25 FAB M5 None -
26 FAB M5 FLT3 p.D839G (0.21) -
    FLT3 p.Y591D (0.08) -
    FLT3 p.D839N (0.07) -
27 FAB M2 KIT p.N822K (0.43) -

aAlterations not reported in Catalogue of Somatic Mutations in Cancer. UPN, unique patient number; AML, acute myeloid leukemia; VAF, variable allele frequency; SNV, single nucleotide variation.

Prognostic genetic alterations at diagnosis according to the European LeukemiaNet guidelines.

UPN Low-risk features Intermediate risk features High-risk features.
1 No KMT2A-MLLT3 Complex karyotype
2 No No No
3 No No Complex karyotype
4 No No No
5 No No Complex karyotype
6 No No FLT3-ITD
7 No No No
8 No No No
9 No No t(6;11)(q27;q23)
10 No t(9;11)(p22;q23) No
11 CEBPA p.Q312HR No No
12 No No No
13 No No Complex karyotype
14 No No Complex karyotype
15 No No TP53 p.K164E
16 PML-RARA No No
17 PML-RARA No No
18 No No KMT2A-MLLT10
19 CBFB-MYH11 No No
20 RUNX1-RUNXT1 No No
21 No No No
22 CBFB-MYH11 No No
23 RUNX1-RUNXT1 No No
24 RUNX1-RUNXT1 No No
25 No No No
26 No KMT2A-MLLT3 No
27 RUNX1-RUNXT1 No No

UPN, unique patient number.