Rev. Bras. Hematol. Hemoter. vol.36 número5

revbrashematolhemoter.2014;36(5):379–380

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

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

Letter

to

the

editor

Detection

of

cytogenetic

abnormalities

in

mature

B-cell

neoplasms:

the

value

of

cultures

with

different

mitogens

Mature B-cell neoplasms are a common group of

hematological malignancies in Western counties with

the most prevalent being chronic lymphocytic leukemia

(CLL).Clonalcytogeneticabnormalitiesareobservedinonly

30% of the CLL cases studied by conventional cytogenetic

techniques(karyotyping)andupto80%ofthecasesstudied

usingthefluorescenceinsituhybridization(FISH)technique

with probes to investigate 6q21,11q22-23 (ATM), 13q14.3

and 17p13 (P53) deletions and for trisomy 12. Cytogenetic

abnormalitiesareassociatedwithprognosis;deletionof13q

is associated with the best prognosis and deletion of 17p

withtheworst.Despite thegreatersensitivityofFISH,it is

expensiveanddoesnotdetectabnormalitiesotherthanthose

investigated. Conventional cytogenetic studies in CLLs are

oftendisappointingbecausenometaphasesoronlynormal

metaphasesareobtained.

Itisveryimportanttosendasamplecontainingthe

neo-plastic cells to be analyzed to the lab for the cytogenetic

study.Inlymphoidmalignanciesthissamplecanbefrom a

lymphnode, thespleen, orother affected tissue,including

bonemarrowandperipheralbloodwhenleukemized.Theuse

ofmitogenicagentstostimulateB-cellsincultureshasbeen

useful.Thepotenttumorpromoter12-myristate 13-acetate

(TPA)hasgiventhehighestclonedetectionrateinthe

cyto-geneticlaboratory.Moreover,culturingisperformedwithout

any other stimulating agent. In the late 1990s, the use of

newcytogeneticprotocolswithdifferentmitogenssuchasthe

CpG-oligonucleotideDSP30plusinterleukin-2(IL-2)improved

the detection ofclonalabnormalities in up to80% in CLL.

DSP30alsoinducestheIL-2alphareceptor(possiblyviaCD25)

inCLLcellstherebyallowingIL-2togenerateaco-stimulatory

effect.

Thecombinationofthesedifferentmitogenswasdescribed

bythreedifferentgroups.

AstudybyDickeretal.inGermany,with132CLLsamples,

showedsuccessfulculturesin125cases,81%ofwhichshowed

clonal abnormalities with the most prevalent being del6q,

del11q,+12,del13q,del17pandthe14q32rearrangement.1

InaBrazilianstudyconductedbyMoratoetal.,clonal

cyto-geneticabnormalitieswere detectedin80%ofthe35cases

studied,showingrecurringabnormalities(del6q,del11q,+12,

del13q, −17/del17q,14q32 rearrangement)aswell as many

others(+4,+5,+8,+11,+15,del12p13,+18,+19,+21,and

bal-ancedtranslocationsinvolvingdifferentchromosomes).2

AstudybyWrenetal.inAustraliafailedtoreproducethese

data. Forty-fivesamples ofCLL weresubmitted tocultures

stimulatedwithTPA,DSP30togetherwithIL-2andcombined

TPAandDSP30+IL-2.Therateofdetectionofchromosomal

abnormalities washigher,significance,inthecultureswith

TPA+DSP30+IL-2(52.9%)thanwithTPA(45.8%)orDSP30+IL-2

(29.2%).Additionally,therewasagreatersuccessintheculture

withTPA(100%)thanwithDSP30+IL-2(72.7%)andthemitotic

indexandresolutionofbandswerealsomoresuccessfulin

theTPAculture.3

In view of these conflicting data in the literature, we

comparedthedetectionofclonalabnormalitiesusingthree

differentculturesmentionedintheliterature:without

stim-ulating agent, TPA and DSP30+IL-2(200U/mL) and another

withahigher concentrationofIL-2(DSP30+IL-2 [400U/mL])

using16samplesofmatureB-cellneoplasms(11wereCLL).

Atleasttenmetaphasesfromeachculturewereanalyzedin

anIkaroskaryotypingSystem(MetaSystems)bytwoanalysts,

and the karyotypewas described accordingtothe

Interna-tional SystemforHumanCytogeneticNomenclature (ISCN)

2013.Table1showstheresultsincaseswithclonal

abnormal-ities.

The incidence of clonal abnormalities in mature B-cell

neoplasms in this study was 50%, with trisomy 12 being

themostcommonabnormality.Theculturewith

DSP30+IL-2 (400U/mL) showed a lower failure rate than that with

DSP30+IL-2(200U/mL).Therewasnodetectionofnewclonal

abnormalities inthe DSP30+IL-2culture compared to TPA.

Theculturewiththeoligonucleotideincreasedreagentcosts

ofkaryotypingby3%withalowerresolutioninG-banding;

however, it was ableto detecta higherpercentage ofcells

380

Table1–Cytogeneticabnormalitiesandpercentageofabnormalclonesincultureswithoutstimulant,withTPA,DSP30

togetherwithIL-2(200U/mL)andDSP30togetherwithIL-2(400U/mL).

Cytogenetic abnormali-ties/neoplasms

Culturewithout stimulant(24and48h)

(%)

CulturewithTPA (72h)

(%)

CulturewithDSP30and IL-2(200U/mL)(72h)

(%)

Culturewith DSP30andIL-2 (400U/mL)(72h)

(%)

−20(CLL) Absenceofmetaphases 20.0 Absenceofmetaphases –

+12(CLL) 0 60.0 Absenceofmetaphases –

+12,del(14)(q22q24)(B cellneoplasms)

83.3 85.7 Absenceofmetaphases –

XXY,der(1)t(1;?)(p13;?), der(3)t(3;?)(p13;?), del(6)(q21)x2,−8,−9, −10,+13,

add(19)(p13.3),−22(B cellneoplasms)

30.0 20.0 – 80.0

+12/del(20)(q11.2)(CLL) 50.0/0 55.5/22.2 – 80.0/0

t(1;13)(q?25;q14), add(17)(p12),−18 (CLL)

10.0 30.0 – 60.0

i(17)(q10)(CLL) 0 20.0 – 50.0

+1,del(1)(p34)(Bcell neoplasms)

Absenceofmetaphases 10.0 – 40.0

TPA:12-myristate13-acetate;oligonucleotide:DSP30;IL-2:interleukin-2.

mitogensmaybeaneffectiveapproachtokaryotypemature B-cellneoplasmsassuggestedbytheAustraliangroup.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

r

e

f

e

r

e

n

c

e

s

1.DickerF,SchnittgerS,HaferlachT,KernW,SchochC. Immunostimulatoryoligonucleotide-inducedmetaphase cytogeneticsdetectchromosomalaberrationsin80%ofCLL patients:astudyof132CLLcaseswithcorrelationtoFISH, IgVHstatus,andCD38expression.Blood.2006;108(9): 3152–60.

2.OliveiraFM,BrandãoDM,Leite-CuevaSD,SimõesBP,IsmaelSJ, FalcãoRP.ImmunostimulatorymetaphaseinductioninCLL: correlationwithG-bandinganalysis,spectralkaryotyping (SKY),FISHandZAP70expression.RevBrasHematolHemoter. 2009;31Suppl.5:5–229.

3.WrenC,MoriartyH,MarsdenK,TeggE.Cytogenetic

investigationsofchroniclymphocyticleukemia.CancerGenet Cytogenet.2010;198(2):155–61.

RobertaMariadaSilvaOliveira∗_{, ElviraDeolindaRodrigues}

PereiraVelloso

HospitalIsraelitaAlbertEinstein(HIAE),SãoPaulo,SP,Brazil

∗_{Correspondingauthorat:Cytogenetics,DepartmentofClinical}

Pathology,HospitalIsraelitaAlbertEinstein(HIAE),Av.Albert

Einstein,627,Morumbi,05652-900SãoPaulo,SP,Brazil.

E-mailaddress:[email protected]

(R.M.daSilvaOliveira).

Received6March2014

Accepted11June2014

Availableonline18July2014

http://dx.doi.org/10.1016/j.bjhh.2014.07.008

1516-8484/©2014Associac¸ãoBrasileiradeHematologia,

HemoterapiaeTerapiaCelular.PublishedbyElsevierEditora