Rev. Bras. Hematol. Hemoter. vol.39 número4

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Revista

Brasileira

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Hematologia

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Hemoterapia

Brazilian

Journal

of

Hematology

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Hemotherapy

Case

Report

Acute

myeloid

leukemia

with

e1a2

BCR-ABL1

fusion

gene:

two

cases

with

peculiar

molecular

and

clinical

presentations

Fernanda

Borges

da

Silva

_,

_João

_Agostinho

_Machado-Neto

_,

Luisa

Corrêa

de

Araujo

Koury

_,

_Virginia

_Helena

_Leira

_Lipoli

_Bertini

_,

Cristina

Alonso

Ratis

_,

_Maria

_de

_Lourdes

_Lopes

_Ferrari

_Chauffaille

_,

Elvira

Deolinda

Rodrigues

Pereira

Velloso

_,

_Belinda

_Pinto

_Simões

_,

Eduardo

Magalhães

Rego

_,

_Fabiola

_Traina

a_{FaculdadedeMedicinadeRibeirãoPreto,UniversidadedeSãoPaulo(FMRPUSP),RibeirãoPreto,SP,Brazil} b_{HospitalIsraelitaAlbertEinstein,SãoPaulo,SP,Brazil}

c_{EscolaPaulistadeMedicinadaUniversidadeFederaldeSãoPaulo(EPMUnifesp),SãoPaulo,SP,Brazil}

d_{HospitaldasClínicasdaFaculdadedeMedicinadaUniversidadedeSãoPaulo(HCFMUSP),SãoPaulo,SP,Brazil}

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Articlehistory:

Received17May2017 Accepted6July2017

Availableonline3August2017

Introduction

Acutemyeloidleukemia(AML)diseaseentitiesareclassified bytheWorldHealthOrganization(WHO)basedonsignificant cytogeneticand moleculargeneticfindings.1,2 _Thecategory of acute myeloid leukemia not otherwise specified (AML-NOS)includescaseswith≥20%myeloblastsintheperipheral

blood(PB)orbonemarrow(BM)intheabsenceofrecurrent geneticabnormalities,myelodysplasia-relatedchangesor pre-viouscytotoxictherapy.CasesofAMLwithdysplasiain≥50%

∗ _{Correspondingauthorat:DepartmentofInternalMedicine,FaculdadedeMedicinadeRibeirãoPretodaUniversidadedeSãoPaulo(FMRP} USP),Av.Bandeirante3900,RibeirãoPreto,SP,Brazil.

E-mailaddress:[email protected](F.Traina).

of the cells in two or more myeloid cell lineages or cases preceded byawell-documented history ofmyelodysplastic syndromes (MDS) or MDS/myeloproliferative neoplasm are definedasAMLwithmyelodysplasia-relatedchanges (AML-MRC).1,2 _{AML-MRC developsin approximately one-third of} MDS andgenerally hasapoorprognosis.3,4 _{Cytogeneticsis} amandatorytoolinthediagnosisofAMLwithchromosome abnormalitiesbeingpresentinapproximatelyhalfoftheadult AML cases; chromosome abnormalities have an important prognosticvalue.5

http://dx.doi.org/10.1016/j.bjhh.2017.07.001

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Metaphase cytogenetics has proven to bean extremely valuable clinical toolin the management ofhematological malignancies.Thismethodology candetect balanced chro-mosomalchanges,includingtranslocationorinversion,and unbalancedchromosomalchanges,includingtrisomy, dupli-cation,anddeletion.Thesuccessofmetaphasecytogenetics requirescellularproliferationandchromosomespreads;the sensitivityand the resolutiondependon the proportionof clonal cells in the tested sample and on the size of the lesion,respectively.6 _{Single nucleotidepolymorphism array} (SNP-A),alsoreferredtoaschromosomalmicroarray,hasbeen appliedasahigh-resolutionwholegenomescanningtoolto detectunbalancedchromosomalchanges.Themajor advan-tageofSNP-Aovermetaphasecytogeneticanalysisisitsability todetecthiddenchromosomaldefects,including submicro-scopic(cryptic)aberrationsandthedistinctionofindividual genotypestodetectcopynumber-neutrallossof heterozygos-ity(CN-LOH),alsodefinedasuniparentaldisomy.7

ThePhiladelphia(Ph)chromosomeresultsfromabalanced translocationt(9;22)(q34;q11.2)thatleadstotheformationof thefusionproteinBCR-ABL1withconstitutivetyrosinekinase activity.ThreedifferentbreakpointclusterregionsintheBCR gene (M-bcr, m-bcr, and ␮-bcr) are reported: 8.5kb hybrid

mRNA(b2a2orb3a2)encodesthe210kDaprotein(p210),7.5kb hybridmRNA(e1a2)encodesthe190kDaprotein(p190)and 9kbhybridmRNA(c3a2)encodesthe230kDaprotein(p230).8 Thet(9;22)(q34;q11.2)isfoundin90–95%ofcasesofchronic myeloid leukemia and around 20% of acute lymphoblastic leukemia.Incontrast,thePhchromosomeisveryrareinAML, accountingforapproximately1–2%ofthecases.9_Neuendorff etal.10_{recentlyreviewedcasereportsandcaseseriesofAML} withBCR-ABL1intheliteraturesince1975,aswellascases fromtheirowninstitution.Theauthorsreportedthatamong the 126confirmed casesofAMLwithBCR-ABL1, 38% were definedbyWHO2008asAML-NOS,32.5%asAML-MRC,and 16.7%ascorebindingfactorleukemia.10

Recently,theWHO2016classificationincludedanew pro-visionalcategoryfordenovoAMLwithBCR-ABL1,recognizing this rare diagnostic entity that may benefit from tyrosine kinaseinhibitortherapy.11_{Thepatientsarestratifiedas} inter-mediateII riskaccording toEuropeanLeukemiaNet and in thepoor-riskgroupaccordingtotheNationalComprehensive CancerNetwork.5,12

Herein,wepresenttwounusualcasesofAMLwith BCR-ABL1. In accordance with WHO 2008 definitions, Case 1 was diagnosed as AML-MRC and presented with complex karyotypeandthee1a2BCR-ABL1genefusion.Case2was diag-nosedasdenovoAML-NOSandpresentedanear-tetraploidy karyotype and the e1a2 BCR-ABL1 gene fusion. Both cases

wereinvestigatedbyconvectionalcytogeneticsandmolecular approaches.

Methods

Patients

Patientsdescribedinthisreportwerefollowedupatthe Hos-pitaldasClínicasoftheUniversidadedeSãoPauloinRibeirão Preto,SãoPaulo,Brazil.TheEthicsCommitteeofthe institu-tionapprovedthestudy,andwritteninformedconsentwas obtained.ThealgorithmproposedbyNeuendorffetal.10_was usedtoexcludethe diagnosisofchronicmyeloidleukemia blastcrisis.

Metaphasecytogenetics

MetaphasecytogeneticswasperformedonBMaspirateusing standardmethodsandthekaryotypewasdescribedaccording totheInternationalSystemforHumanCytogenetic Nomen-clature(ISCN)2013.13

Singlenucleotidepolymorphismarray

FortheSNP-A,genomicDNAwasextractedfromBM accord-ing to the manufacturer’s instructions (QIAGEN DNA Kit, Valencia,CA,USA). SNP-AwasperformedusingAffymetrix Genome-Wide HumanSNPCytoscanHD (Affymetrix,Santa Clara,CA,USA).ThefileswereanalyzedusingChromosome AnalysisSuite(ChAS)software.Regionsofcopynumber vari-ants(CNVs)largerthan1MbandCN-LOHlargerthan10Mb were denoted as true aberrations. In order to detect the somaticorigin copy number alterationsdistinguishedfrom constitutionalpolymorphicCNVs,thelesionsidentifiedusing SNP-A were comparedwiththeDatabaseofGenomic Vari-ants(DGV;http://projects.tcag.ca/variation).Aberrationsthat were identifiedbySNP-Aweredescribed accordingtoISCN 2013.13

Reversetranscriptase-polymerasechainreactiontodetect BCR-ABL1

TotalRNA(1␮g) wasobtainedfrom thepatient’speripheral

bloodcellsandsubmittedtoreversetranscriptionpolymerase chainreaction(RT-PCR)usingtheHighCapacitycDNAReverse TranscriptionKit(LifeTechnologies,Carlsbad,CA,USA).The first PCRwas performed on avolume of 25␮L containing:

1× reaction buffer, 1.5mM MgCl₂, 200␮M dNTPs, 10pmol

of each primer (Mbcr1: 5′

-GAAGTGTTTCAGAAGCTTCTC-3′_{; 2oabl1: 5}′_{-TGATTAAGCCTAAGACCC GGA-3}′_{; mbcr1: 5}′

-CCATCGTGGGCGTCCGCA-3′_{),0.5UTaqDNApolymerase(Life}

Technologies) and 1.5␮L cDNA. The first PCR conditions

were an initial phase of 2min at 94◦_{C, then 25 cycles}

at 94◦_{C for 30s, 51}◦_{C for 40s, 72}◦_{C for 1min, followed}

by a final extension step at 72◦_{C for 7min. The second}

PCR was performed on a volume of 25␮L containing: 1×

reaction buffer, 1.5mM MgCl2, 200␮M dNTPs, 10pmol of

each primer (Mbcr2: 5′

-TGGAGCTGCAGATGCTGACCAACTC-3′_;mbcr2:5′_{-AGATCTGGCCCAACGATGGCGAGGGC-3}′_;2iabl2:

5′_{-ATCTCCAGTGGCCAGAAAATCATAC-3}′_),0.5UTaqDNA

poly-merase(LifeTechnologies)and1␮LofPCRproductsfromthe

firstreaction.SecondPCRconditionswereaninitialphaseof 2minat94◦_{C,then35cyclesat94}◦_Cfor30s,60◦_Cfor30s,72◦_C

for1min,followedbyafinalextensionstepat72◦_Cfor7min.

ThePCRproductswereanalyzedon2%agarosegelstained withethidiumbromide.

Case

1

An 82-year-old male with systemic hypertension, with-out other relevant medical history and good performance status(Easterncooperativeoncologygroup: 0)sought med-ical attention complaining of weight loss, asthenia, and petechiaeduringtheprevious45days.Physicalexamination revealed pallor, petechiae and absence of hepatomegaly andsplenomegaly.Thecomplete blood countshowed pan-cytopenia (hemoglobin 7.2g/dL, white blood cell count 2.5×109/L, neutrophil count 0.3×109/L, lymphocyte count

2.2×109/L, and platelet count 8×109/L). BM aspiration

revealed20%myeloblasts,anddysplasticalterationsin70% of erythroid and megakaryocytic lineages (Figure 1A–F). The immunophenotypic analysis showed that the blasts were positive for human leukocyte antigens – (HLA)-DR, CD13,CD33,CD34,CD117,CD7, CD133,and CD56and neg-ative for CD19, CD15, CD11c, CD42a, CD22, CD2, CD11b, CD64, CD14, and CD36. The patient was started on sub-cutaneous low dose cytarabine immediately after the diagnosisofAML.Metaphasecytogeneticsanalysisrevealed 46,XY,t(9;22)(q34;q11),del(20)(q11)[2]/46,idem,inv(7)(q22q36) [6]/47,idem,inv(7)(q22q36),+19[12] (Figure 1G–I). An RT-PCR assay for BCR-ABL1 confirmed the presence of t(9;22) and revealedap190BCR-ABL1isoform(e1a2BCR-ABL1)(Figure1J). The SNP-A identified the loss of 2q36 not identified by metaphase cytogenetics: arr[hg19] 2q36.3q37.3(229,385,664-239,698,667)x1, and confirmed the loss of 20q11: arr[hg19] 20q11.21q13.13(30,955,339-49,197,975)x1. As expected, the SNP-Adidnotdetectthebalancedtranslocationt(9;22), nei-thertheinversionof7q.SNP-Aallowedfortheidentification ofthelossof7q36notidentifiedbymetaphasecytogenetics: arr[hg19]7q36.3(155,607,058-156,777,458)x1.However,SNP-A failed to identifythe trisomy ofchromosome 19, probably explained bythe limited sensitivity ofthe test forlesions presentinlessthan30%oftheunculturedcells(Figure1K). ThepatientwasclassifiedasAMLwithBCR-ABL1according toWHO2016andasAML-MRCaccordingtoWHO2008.The patient presented persistent pancytopenia and increased BM blasts following two cycles of subcutaneous low dose

cytarabine and was started on imatinib. No hematologic responsewasobservedaftertwomonthsofimatinibandthe patientsubsequentlydiedofinfectiouscomplications.

Case

2

A 28-year-old woman presented with leukocytosis (white blood cell count47.8×109/L, myeloblasts 66%,

metamyelo-cytes 1%, neutrophil band 1%, segmented neutrophils 4%, lymphocytes28%),anemia(hemoglobin6.9g/dL),and throm-bocytopenia(33×109/L).Thepatienthadbeensufferingfrom

weakness and fatigue associated with febrile episodes for onemonth.Therewasnopastmedicalhistoryofany hema-tologicaldisorder.Physicalexaminationrevealed pallorand absence ofhepatomegaly andsplenomegaly. BMaspiration showedmyeloblastscomprising88%ofallcells(Figure2A–C). Theimmunophenotypicanalysisshowedthattheblastswere positiveforCD34,CD117,CD33,CD13,HLA-DR,CD38,CD11b, CD4, CD133, CD15,CD11c and CD64and negativefor CD7, CD56,CD19,CD41andNG2.Thepatientwasdiagnosedwith

de novo AML-NOS and classified as acute myelomonocytic leukemia according to WHO 2008 or AML with BCR-ABL1 according to WHO 2016. The cytogenetic analysis showed 92<4n>,XXXX,−4,−5,+6,−7,−11,+12,−13,−14,+15,−17,−21,

t(9;22)(q34;q11)x2[cp20] (Figure 2D) and a RT-PCR assay for

BCR-ABL1revealedap190isoform(e1a2BCR-ABL1)(Figure2E). The patient received two cycles of inductiontherapy with daunorubicin and cytarabine, associated with intrathecal chemotherapy (methotrexate, cytarabine and dexametha-sone).Atthesecondcycleofinduction,thepatientwasstarted on dasatinib. Thepatient achievedcomplete hematological remissionandreceivedmatchedrelatedmyeloablative allo-geneichematopoieticstemcelltransplantation.Atthetime ofthisreport,twentymonthsafterdiagnosis,sheremainsin completeremission.

Discussion

Herein,wedescribedthecharacteristicsoftwocasesofAML withBCR-ABL1. Althoughveryrare, thepresenceofthePh chromosomehasbeenreportedinsomeMDScasesat diagno-sisoratthetimeofdiseaseprogression,andbothBCR-ABL1

variants,p210andp190,havebeenpreviouslydescribed.9,14,15 Inacohortcontaining148patientswiththePhchromosome, Keungetal.9_{reportedonlytwocasesofdenovoAML(1%)and} threecasesofMDS(2%).Fukunagaetal.14_{reportedacaseof} MDSthatpresentedanabruptevolutiontoAML-MRCandthe acquisition ofthe Phchromosome(p210BCR-ABL1).Inthat report, thepatienthadhematological responsetonilotinib, whichwaslostwiththeacquisitionoftheBCR-ABL1Y253H mutation. Subsequently,thepatient hadtransient hemato-logicalresponsetodasatinib,butresponsewaslostwiththe acquisitionoftheBCR-ABL1T315IandE255Kmutations.

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Figure2–Bonemarrowmorphological,cytogeneticsandmolecularanalysisofCase2.(A)Hypercellularbonemarrow fragment.(B)ThebonemarrowwasstainedbyWright-Giemsastainandahighfrequencyofmyeloblastswasobserved.(C) Thebonemarrowwasstainedbymyeloperoxidasestain.(D)Illustrativemetaphase:91<4n>,XXXX,−5,t(9;22)(q34;q11)x2.

Thet(9;22)(q34;q11.2)isindicatedwithbluearrows.(E)Polymerasechainreactionassayforb2a2/b3a2ande1a2BCR-ABL1

transcripts–1:ladder50basepairs(LifeTechnologies);2:Case#2;3:positivecontrolforb2a2/b3a2BCR-ABL1_transcript

(p210);4:positivecontrolfore1a2BCR-ABL1_{transcript(p190);5:negativecontrolforb2a2/b3a2ande1a2}BCR-ABL1

transcripts.

chromosomebandisinvolvedinmostcaseswith7qdeletions inMDSandAML.16,17_{Deletionofthelongarmofchromosome} 20isacommonrecurringchromosomalabnormality associ-atedwithmyeloidmalignancies.18

In AML, the near-tetraploidy karyotype is a rare cyto-genetic alteration and its prognostic relevance is not well defined.19 _{Yamaguchi et al.}15 _{reported a case of AML} withBCR-ABL1whopresentedmetaphasecytogeneticswith 92,XXYY,t(9;22)(q34;q11)x2[20]andRT-PCRpositivityforp190

BCR-ABL1;thepatientachievedcompleteremissionafterthe firstcycleofinductionchemotherapyandwasconsolidated withallogeneichematopoieticstemcelltransplantation.15

Inconclusion,cytogeneticabnormalitiesidentifiedby kary-otypingremainanimportantprognosticfactorinAML.The report of these rare cases of AML with the presence of e2a1BCR-ABL1 and futureanalyses forthesealterations in AMLpatientsareimportanttodefinethelocalincidence,to improvediagnosisandclassification,andtobetterassessthe impactofthesemolecularalterationsontheoutcomesofAML patients.

Funding

ThisworkreceivedfinancialsupportfromConselhoNacional de Desenvolvimento Científico e Tecnológico (CNPq) and

Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors would like to thank Andy Cumming for the English review,and Amélia Góes and ClaudiaHelena Mag-nanifortheirvaluabletechnicalassistancewithRT-PCRfor BCR-ABL1detection.

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