Rev. Bras. Hematol. Hemoter. vol.37 número3

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rev bras hematol hemoter. 2015;37(3):211–214

w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Letter

to

the

Editor

Somatic

mutations

of

calreticulin

in

a

Brazilian

cohort

of

patients

with

myeloproliferative

neoplasms

DearEditor,

Essentialthrombocythemia(ET),polycythemiavera(PV),and primarymyelofibrosis (PMF) are Philadelphia chromosome-negativemyeloproliferativeneoplasms(MPNs)characterized by increased myeloid proliferation. The gain of function inducedbythe Januskinase2mutation,JAK2V617F_,_has_been

reportedinmostPV andinmorethan halfofETand PMF cases.1_However,_the_presence_of_different_disease_phenotypes

andtheabsenceoftheJAK2mutationinsomeMPNssuggests thatadditionalgeneticlesionsor/andaberrantsignaling path-waysmaybeinvolvedinthepathogenesisofthesediseases.1,2

In December 2013, somatic mutations inthe calreticulin

(CALR)genewere identifiedinETand PMFpatients bytwo independent groups3,4 _and _confirmed _by _others.5–9 _CALR

mutations have been reported as mutually exclusive with

JAK2and MPLmutationsandmay bepresent in56–88%of

JAK2/MPL-negativecases.3,4_A_recent_paper_reported_a_patient

thathadbothmutations,JAK2V617F_and_a_CALR_exon₉

muta-tion,simultaneously.10_Over_thirty_different_CALR_mutations_in

exon9havebeendescribed,butthemostfrequentmutations (about80%)maybeclassifiedastype-1[L367fs*46;deletionof 52basepairs(bp)]andtype-2(K385fs*47;insertionof5bp).3,4

Thefunctionalchangesinducedbythemutationsarestillnot completelyelucidated,buttheoverexpressionofthetype1

CALRmutationinBa/F3cells(anIL3dependentcellline)leads tocytokine-independentcellgrowthand STAT5activation.4

GeneexpressionsignaturestudiesalsoindicatethatJAK2and

CALR mutations share mechanisms ofmalignant transfor-mation,reaffirmingacentralroleoftheJAK/STATsignaling pathwayinthepathogenesisofMPN.11

The aim of the present study was to characterize the prevalenceofCALR mutationsand the clinical and labora-torialcharacteristicsofCALR-mutatedpatientsinaBrazilian cohortofMPNs.Seventy-threeMPNpatientswereincludedin thestudy(ET=32,PV=20,PMF=21).Patients’characteristics aredescribedinTable1.Peripheralbloodsampleswere col-lected,submittedtohemolysis,andDNAwasextractedbythe

phenol/chloroformmethod.Allsampleswereinvestigatedfor

JAK2andCALRmutations.TheJAK2V617F_mutation_and_CALR

exon9mutationswereverifiedaspreviouslydescribed.12,13 CALRmutationswereclassifiedastype-1(deletionof52bp), type-2(insertionof5bp)orothers(Figure1).

Inourcohort,CALRmutationswerefoundin20patients

(13ETand7PMF;Table1)andweremutuallyexclusivewith

JAK2V617F_:_20/73_(27%)_of_total_MPN_patients_and_20/28_(71%)

oftheJAK2WT_patients. _Among_the_CALR_-mutated_patients,

Type-1CALRmutationswerefoundin50%(10/20;8ETand2 PMF),type-2in40%(8/20;4ETand4PMF),andothersin10% (1ETand1PMF)oftheindividuals.CALRmutationswerenot detectedinPVpatients(allJAK2V617F_positive)._In_ET,_CALRMUT

patientsshowedreduced hemoglobinlevelscomparedwith

JAK2V617Fpatients(p-value<0.01;Table2);nodifferenceswere observedinwhitebloodcell,neutrophilandplateletcounts, thromboticevents,hepatomegaly,splenomegalyand consti-tutionalsymptoms.InPMFpatients,CALRmutationalstatus wasnotassociatedwithclinicalfeatures(Table2).The fre-quencyofCALRmutationsinthisBraziliancohortwassimilar to previously described frequencies.3,4,7 _Furthermore,_CALR

andJAK2V617F_mutations_were_mutually_exclusive._Knowledge

regardingtheclinicalimpactofCALRmutationsinMPNsis stillunderconstruction,butsomestudiesindicatethatCALR -mutated patients have lower ages at disease onset, lower hemoglobinandplateletcounts,andhavebetteroverall sur-vivalthaneitherJAK2-mutatedorCALR/JAK2/MPLwild-type patients.5,9,14_In_our_cohort, _CALRMUT_ET_patients_presented

lowerhemoglobinlevels,comparedwithJAK2V617F_ET_patients,

even though in both groups hemoglobin values remained withinthereferencerange.

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revbrashematolhemoter.2 0 1 5;37(3):211–214

Table1–Patientcharacteristics.

MPN(n=73) ET(n=32) PV(n=20) PMF(n=21)

Age(years)–median(range): 57(19–87) 53(19–83) 70(41–88) 62(27–87)

Gender–male/female 30/43 10/22 9/11 11/10

Hemoglobin(g/dL)–median(range) 13.9(8.1–22.7) 13.6(9.7–17) 18.4(12.8–22.7) 12.5(8.1–19.9) WBCcount(×109L–1)–median(range) 9.8(2.4–43.2) 9(3.0–17.8) 11.2(6.3–25.5) 11.8(2.4–43.2)

Plateletcount(×109L–1)–median(range) 764(108–2065) 914(541–2065) 609(203–1070) 661(108–1716)

JAK2mutationstatus–n(%)

JAK2WT ₂₈₍₃₈₎ ₂₀₍₆₃₎ ₀₍₀₎ ₈₍₃₈₎

JAK2V617F ₄₅₍₆₂₎ ₁₂₍₃₇₎ ₂₀₍₁₀₀₎ ₁₃₍₆₂₎

CALRmutationstatus–n(%)

CALRWT ₅₃₍₇₃₎ ₁₉₍₅₉₎ ₂₀₍₁₀₀₎ ₁₄₍₆₇₎

CALRMUT ₂₀₍₂₇₎ ₁₃₍₄₁₎ ₀₍₀₎ ₇₍₃₃₎

MPN:myeloproliferativeneoplasms;ET:essentialthrombocythemia;PV:polycythemiavera;PMF:primarymyelofibrosis;WBC:whitebloodcell count;JAK2:Januskinase2gene;WT:wild-type;CALR:Calreticulingene;MUT:exon9mutations.

20000

245 255 265 275 285 295 305 315 230 240 250 260 270 280290300310 250 260 270 280 290 300 240 260 280 300

302 bp 297 bp 291 bp 245 bp

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A

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M NTC #1 #2 #3 #4 #5 #6 #7

Type-1

Type-1 Type-2 Other

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MPN patients

Calreticulin

_mutations

Calreticulin mutations

Other

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Figure1–Calreticulin(CALR)indelmutationsinmyeloproliferativeneoplasm(MPN)patients.(A)PCRamplificationofexon9 ofCALRgeneloadedon4%agarosegel;lane1:100bpmarker(M);lane2:notemplatecontrol(NTC);MPNpatients#1and#2:

CALRWT_;_#3_and_#4:_{heterozygotes}_for_type-1_CALR_mutation₍_CALRWT_amplicons:₂₉₇_bp;_type-1_CALRMUT_amplicons:₂₄₅_bp);

#5and#6:heterozygotesfortype-2CALRmutation(CALRWT_amplicons:_297;_type-2_CALRMUT_amplicons:₃₀₂_bp);_#7:

heterozygotefornon-type-1or-2CALRmutation(CALRWT_amplicons:₂₉₇_bp;_CALRMUT_amplicons:₂₉₁_bp)._(B)

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T able 2 – Clinical and labor a tory fea tur es of essential thr omboc ythemia and pr imary m y elofibr osis pa tients, str a tified accor ding to JAK2 and C ALR m uta tional sta tus. Essential thr omboc ythemia Primar y m y elofibr osis b C ALR MUT JAK2 V617F C ALR WT / JAK2 WT p -v alue a , c p -v alue a , d C ALR MUT JAK2 V617F p -v alue a , c Gender: male/female – n (%) 4 (31)/9 (69) 5 (42)/7 (58) 1 (14)/6 (86) 0.69 0.61 4 (57)/3 (43) 6 (46)/7 (54) 1.00 Ag e – y ears (r ang e) 57 (20–82) 55 (44–83) 43 (19–53) 0.61 0.13 53 (27–80) 65 (43–87) 0.30 Hemo globin – g/L (r ang e) 13 (10–15) 14 (12–17) 14 (12–16) 0.007 0.12 12 (9–13) 14 (8–20) 0.08 White b lood cells – × 10 9L –1 (r ang e) 7.1 (3–17.8) 9.2 (4.5–16.7) 9 (5–11.8) 0.64 0.69 9.4 (2.4–35.7) 11.8 (8.4–43.2) 0.38 Neutr ophils – × 10 9/L (r ang e) 4.8 (2.1–15.1) 6 (2.5–12) 5.6 (3.2–8.5) 0.43 0.48 6.2 (1–22.5) 7.1 (0.5–38.3) 0.48 Platelets – × 10 9/L (r ang e) 938 (593–2065) 880 (541–1340) 984 (617–1274) 0.37 0.87 567 (108–1716) 661 (129–1606) 0.69 Thr ombotic ev ents – n (%) 0 (0) 2 (17) 0 (0) 0.50 1.00 0 (0) 1 (8) 1.00 He patome g al y – n (%) 1 (8) 0 (0) 0 (0) 1.00 1.00 0 (0) 2 (17) 1.00 Splenome g al y – n (%) 2 (15) 1 (8) 1 (14) 1.00 1.00 5 (71) 7 (54) 0.64 Constitutional symptoms – n (%) 1 (8) 3 (25) 2 (29) 0.32 0.27 1 (14) 3 (23) 1.00 a F isher’ s e xact test wa s used for cate gorical factors with 2 le v els; Mann–Whitne y test for measur ed factors. b Onl y one patient with primar y m y elofibr osis pr esented C ALR WT / JAK2 WT . c C ALR MUT vs. JAK2 V617F . d C ALR MUT vs. C ALR WT / JAK2 WT .

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

r

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f

e

r

e

n

c

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s

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JoãoAgostinhoMachado-Neto1_,_Paula_de_Melo_Campos1_,

DulcinéiaMartinsdeAlbuquerque,FernandoFerreiraCosta, IreneLorand-Metze,SaraTerezinhaOlallaSaad,

FabiolaTraina∗

UniversidadeEstadualdeCampinas(UNICAMP),Campinas,SP, Brazil

∗_{Corresponding}_author_at_: _Rua _Carlos_Chagas, _480,_13083-878

Campinas,SP,Brazil.

E-mailaddresses:ftraina@fmrp.usp.br,

fabiolatraina@gmail.com(F.Traina).

1_Both_authors_contributed_equally_to_this_work.

Received19September2014 Accepted25February2015 Availableonline15April2015

http://dx.doi.org/10.1016/j.bjhh.2015.03.012