w w w . h t c t . c o m . b r
Hematology, Transfusion and Cell Therapy
Review article
Detection of somatic TP53 mutations and 17p deletions in patients with chronic lymphocytic leukemia: a review of the current methods
Maria de Lourdes L.F. Chauffaille
a,∗, Ilana Zalcberg
b,c, Wolney Gois Barreto
d, Israel Bendit
eaGrupoFleury,SãoPaulo,SP,Brazil
bCentrodeTransplantedeMedulaÓssea,InstitutoNacionaldoCancer(CEMO-INCA),RiodeJaneiro,RJ,Brazil
cGeneOne,DASA,SãoPaulo,SP,Brazil
dHemocentro,RibeirãoPreto,SP,Brazil
eLaboratóriodeBiologiadoTumordoServic¸odeHematologiadoHospitaldasClínicasdaFaculdadedeMedicinadaUniversidadedeSão Paulo(FMUSP),SãoPaulo,SP,Brazil
a r t i c l e i n f o
Articlehistory:
Received13December2019 Accepted21May2020 Availableonline25June2020
Keywords:
Chroniclymphocyticleukemia TP53mutations
17pdeletions
a bs t r a c t
Chroniclymphocyticleukemiaisthemostcommonhematologicmalignancyamongadults inWesterncountries.SeveralstudiesshowthatsomaticmutationsintheTP53geneare presentinupto50%ofpatientswithrelapsedorrefractorychroniclymphocyticleukemia.
ThisstudyaimstoreviewandcomparethemethodsusedtodetectsomaticTP53mutations and/or17pdeletionsandanalyzetheirimportanceinthechroniclymphocyticleukemia diagnosisandfollow-up.Inchroniclymphocyticleukemiapatientswithrefractoryorrecur- rentdisease,theprobabilityofclonalexpansionofcellswiththeTP53mutationand/or 17pdeletionisveryhigh.Thestudiesassessedshowedseveralmethodologiesabletodetect thesechanges.Forthe17pdeletion,thechromosomeG-banding(karyotype)andinterphase fluorescenceinsituhybridizationarethemostsensitive.Forsomaticmutationsinvolving theTP53gene,moderateor high-coveragereadnext-generationsequencingandSanger sequencingarethemostrecommendedones.TheTP53genemutationsrepresentastrong adverseprognosticfactorforpatientsurvivalandtreatmentresistanceinchroniclympho- cyticleukemia.Patientscarryinglow-proportionTP53mutation(lessthan20–25%ofall alleles)remainachallengetothesetests. Thus,foranyofthemethodsemployed,itis essentialthatthelaboratoryconductitsanalyticalvalidation,documentingitsaccuracy, precisionandsensitivity/limitofdetection.
©2020Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).
∗ Correspondingauthorat:Av.WaldomirodeLima508,SãoPaulo,CEP:04344-070SP,Brazil.
E-mailaddress:mlourdes.chauffaille@grupofleury.com.br(M.L.Chauffaille).
https://doi.org/10.1016/j.htct.2020.05.005
2531-1379/©2020Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Chronic lymphocytic leukemia (CLL) is the most frequent hematologicmalignancyamongadultsinWesterncountries.
Itaffectsmainly theelderly population,being rare inindi- viduals aged<40 years.Most patientslive 5–10years from diagnosis,whilesomedieearlierduetocomplicationsfrom thedisease.1,2
CLLischaracterizedbyaprogressiveaccumulationofCD5+ B-cell lymphocytes in the peripheral blood, lymph nodes, spleenandbone marrow.Thedisease isextremely hetero- geneousfrom bothabiologicalandaclinicalpointofview.
Thediagnosisisbasedmainlyonthecompletebloodcount (CBC) and the morphological evaluation of a blood smear, whichshowleukocytosiswithlymphocytosis(>5000/L)and onimmunophenotyping oftheperipheralblood circulating B-cells,whichidentifies aclonalB-cellpopulationwiththe CD5marker,inadditiontoB-cellmarkers(CD19,CD20,CD22 andCD23).3,4Ofnote,theBrazilianGroupofFlowCytometry (GBCFLUX)consensusprovidesimportantrecommendations forthediagnosis ofCLL.4–6 Theriskishigher amongmen, witha ratioof 1.7menaffected foreach woman withthe disease.7 TheincidenceofCLLintheUSbetween2006and 2010was 4.2 casesper 100,000 individuals, consideringall ages.8Amongindividualsaged<65years,theincidencewas 1.3/100,000,increasingto24.8/100,000inthepopulationaged
>65years.8InBrazil,therearenoestimatesonthe specific incidenceofCLL. Consideringall leukemias combined,the estimatefor2016is4.4casesper100,000womenand5.6cases per100,000men.9
The CLL treatment has substantially evolved in recent decades.Themediansurvivalincreasedfrom5to7yearsin the70sto10–12yearsinthepresentday.10Inarecentpub- licationbytheBrazilianRegistryofCLL,whichincludeddata from1903patients,theoverallsurvivalwas88%at3yearsand 82%at5years.11Oneofthepillarsinthisevolutionwasthe identificationofmolecularchangesassociatedwithresponse totherapy.Thesechangesarethemainprognosticfactorsof overallsurvival.10,12
Mutations in the immunoglobulin heavy chain variable region(IGHV) genesand mutationsand/ordeletions inthe TP53 gene, mapped in 17p, are among the most relevant molecularsomaticchanges inCLLresearchand treatment.
Recently,aninternationalindexforprognosisofthedisease wasdeveloped,theChronicLymphocyticLeukemia-Interna- tionalPrognosticIndex(CLL-IPI),whichincludesthesegenetic alterations, serum 2-microglobulin levels, patientage and clinicalstageaccordingtotheRaiandBinetclassifications.13
Del17p/TP53alterationsarethemostimportantprognos- ticandpredictivemarkersfortreatmentdecisionsinCLL.14 They have been shown to convey resistance to standard chemo(immuno)therapies, such as fludarabine, cyclophos- phamideandrituximab.Inaclinicaltrial(CLL4)thatcompared first-line treatment with chlorambucil versus fludarabine, withorwithoutcyclophosphamide,progression-freesurvival (PFS)and overall survival(OS)were significantly shorterin TP53mutpatientsthaninpatientswithwild-typeTP53,inde- pendent of the allocation group.15 In the CLL8 trial, that evaluatedfirst-linetherapywithfludarabineandcyclophos-
phamide (FC) or FC with rituximab (FCR) among patients with untreatedCLL,TP53 alterations alsohad a significant negativeimpactonboththeOSandPFS.16Similarly,thecom- plexkaryotypepredictedthelackofresponsetochlorambucil andrituximabasfirst-lineinductiontreatment,withorwith- out rituximabas maintenance,inastudy withelderly CLL patients.17Untilafewyearsago,alemtuzumabandallogeneic stemcelltransplantationweretheonlyeffectivetreatments for those patients. Recently, ibrutinib, idelalisib and vene- toclax,whichinhibitstheBrutontyrosinekinase(BTK),the phosphatidylinositol 3-kinase(PI3K) and BCL2, respectively, havebeen approved,based ontheirefficacy inCLL clinical trialswithpatientsharboringdel17p/TP53mutations.10
The p53 protein is a crucial transcription factor for the cell because it controls several biological processes, such as cell cycle, differentiation, DNA repair, apoptosis and angiogenesis.18 Defects in the p53 pathway prevent DNA repairand apoptosis, resulting in genomic instability and abnormalcell proliferation.18Several studiesanalyzing sequentialsamplesfromCLLpatientsdocumentedtheappar- entacquisitionof17pdeletionsandTP53mutationsandthe expansionofthecellpopulationwiththesechanges(clonal expansion).Accordingtothesestudies,abnormalitiesinTP53 are present in up to 50% of the patients with relapsed or refractorydiseaseandonly5–15%ofnewlydiagnosedcases orimmediatelybeforethefirsttreatment.15,16,19–22
In 2015, Landauet al. published a study that observed a concordancebetween the increase ofdel(17p) and small mutationsinTP53aftertreatmentandrelapseinall12cases of CLL presenting the twotypes ofevent; furthermore, no newTP53mutationwasdetectedinanyofthemovertime.
Basedontheseand otherresultsand apaperpublishedin thejournalCellin2013byDavolietal.theauthorssuggested that theTP53 geneisoneofthefew tumorsuppressorsin whichtheinactivationofthetwoalleles(thefamoustwo-hit hypothesisproposedbyKnudson)isreallynecessaryforclonal expansion.23SimilarresultswerepublishedbyAminetal.in 2016.24
Recentstudies,basedon highsensitivitymethods,have detected TP53 mutationsin subclones. These studies have shownthattheproportionofCLLcaseswithTP53mutationsat diagnosis/treatmentinitiationismuchhigherthanpreviously thoughtandthat,evenatthesubclonelevel,suchmutations mayconferresistancetoclonaltreatmentandexpansion.25–27 Basedonthesefindings,mostmedicalsocieties,includingthe BrazilianGroupofCLL,recommendtheinvestigationof17p deletionsandTP53mutationsbeforeinitiatingfirst-linetreat- mentoranyfurtherlines.4
The objective of this study was to conduct a reviewof theteststodetectsomaticTP53mutationsand17pdeletions andthereafteranalyzetheirimportanceforthediagnosisand long-termfollow-upofCLL.
Testsfordetectionof17pdeletions
FISHandkaryotype
The FISH is a sensitive and quantitative method for the detectionofdel(17p)andisthemethodofchoiceofmostlab- oratoriestodate.Itiswellstandardizedandreproducible.28 Importantly,thecut-off valueforthepercentageofdel(17p)
cells that predict poorer outcomes, regarding overall sur- vivaland progression-freesurvival,hasbeenestablishedas 10–20%,avaluethatisundoubtedlywithintheFISHlimitof detection.29–31
TheFISHanalysisis recommendedbeforethe initiation oftherapyforprogressiveCLLandismandatoryincasesof chemotherapy withalkylating agentsor purine analogues.
GiventheriskofclonalexpansioninCLLpatients,theFISH shouldberepeatedatleastbeforeeachnewtreatmentinall patientswithnodeletiondetectedinaprevioussample,espe- ciallyinthosewithtreatment-refractoryCLLorwhoshowan aggressiveclinicalcourse.4,32,33
OnelimitationoftheFISHisthatitcannotdetecttheso- calledcomplexkaryotype,definedasthepresenceofmultiple cytogenetic, numerical and/or structuralchanges. Conven- tionalcytogenetics(karyotyping)candetectthosealterations andshouldbeperformed,therefore,alongwiththeFISH.The complex karyotype is also associated witha poor progno- sisinCLL.34 Inarecentpaper,Herlingetal.35analyzed161 CLL patients bychromosomeG-banding in stimulated cul- tures,FISHandNGSforapanelof85genesfromperipheral bloodsamples.Thekaryotypeanalysisrevealedthat69%of thepatientspresentedchromosomalalterations,31%,translo- cationsand20%,thecomplexkaryotype.Theyalsofoundthat 76%ofthepatientshadmutationsinoneofthepanelgenes, includingtheTP53.Aparticularlynoteworthyfindingwasthat thecomplexkaryotypeandchangesinvolvingTP53(mutations ordeletions)actedasindependentprognosticfactors.
Othermethods
Othermethodsthatcanbeusedtodetectdel(17p)andother deletionsandinsertionsare thesingle-nucleotide polymor- phism(SNP)array,comparativegenomichybridization(CGH) arrayand multiplexligation-dependent probeamplification (MLPA).Ofnote,studiesperformedtodatesuggestthatthese methods have lower sensitivity, when compared with the FISH,failingtodetectclonessmallerthan20–30%ofthetotal cells.32,36
Apromisingmethodtoidentifysubclonesthatcorrespond toatleast10%ofthetotalBcellsistheNGS.Althoughcurrent CLLtreatmentalgorithmsstilldonotincorporaterecommen- dationsforthedetectionofsubclones,researcheshaveshown thatsubclonalTP53mutationsandthedel17pareassociated withoverallsurvivalratesasshortasthoseobservedinCLL patientswithclonaldefects.27,37Asthedetectionoflargedele- tions andduplications hasbeenthe object ofoptimization inseveral studies on the NGS, eventually this should also becomethe methodologyofchoiceforthedetection ofthe del(17p).38–40
TestsfordetectionofsomaticTP53mutations
Basedonitspredictivevalue,allcurrenttreatmentguidelines forCLLrecommendpriortestingforTP53mutations.41–43The testingshould berepeatedwitheach newdisease progres- siondemandingtreatmentbecauseoftheexpansionofaltered clones.37,42
Specificmethodsandconditionsfordetectingsmallmuta- tionsintheTP53varysignificantlybetweenlaboratories.To reducethisvariability,theEuropeanResearchInitiativeonCLL
(ERIC),44 publishedin 2012,indicatedthe mostappropriate methodsfordetectingTP53mutationsandthecriteriaforthe analysis42:Sangersequencing,denaturinghigh-performance liquid chromatography(DHPLC),functional analysisofsep- arated alleles inyeast (FASAY), arrays and NGS. Given the increased sensitivity of the NGS approach, the ERIC has recentlyupdateditsrecommendationsfortheTP53mutation analysisinCLL,buthasalsoadvisedthatpre-screeningmeth- ods,suchastheDHPLC,arestillcost-effective.45
For cases in which the proportion of leukemic cells in peripheral blood is less than 60%, the ERIC recommended theuseofbonemarrow,lymphnodesorCD19+cellselection beforethetest,especiallyfortheSangersequencinganalysis, whosesensitivityisusuallylowerthantheothermethods.The guidelinesalsoreinforcedthat,formostmethodsconsidered, anewPCRreactionfollowedbySangersequencingshouldbe conductedtoconfirmthemutations.42,45
Arrays
Two array platforms are most commonly used: GeneChip p53 from Affymetrixor AmpliChipp53 from Roche.46 Both arebasedonDNAhybridizationwitholigonucleotideprobes immobilizedonachip andaplatformbased ontheexten- sionofprimersconnectedtothearray,usingthetestedDNA asatemplate.Inthefirstcase,eachmutatedbaseisrepre- sented byatleasttwoprobes,wildand mutated.TheDNA ismarkedwithfluorescencebeforethehybridizationandthe signalintensityofeachoftheprobesischeckedfortheanaly- sisoftheresult.Themethodbasedontheextensionofprimers connectedtothearrayhasasaprincipletheincorporation ofoneoffourdideoxynucleotidesmarkedwithdifferentcol- ors(fluorophores).Theprimerattachedtothearrayisabase sequence ofthe TP53 gene that terminatesjust before the queried(potentiallymutated)base.ThePCRproductsobtained fromtheDNAarehybridizedwiththearrayandthebaseadded totheimmobilizedprimeriscomplementarytotheDNA.47
The AmpliChip, resulted from the development of the GeneChip, which was the first generation ofchips. It was designedtodetectallsingle-basesubstitutionsanddeletions ofasinglenucleotideinexons2–11oftheTP53geneatthe respectivesplicesitesandtoachieveadetectionlimitof25%of mutatedalleles.42,46Theprimerextensionarraywasdesigned todetect95%oftheknownmutationsinexons2–9oftheTP53 gene.42,47Themainadvantagesarethespeedandpracticality ofthetests.Moreover,theydonotrequireextensiveoptimiza- tion.Inadditiontothecost(ofequipmentand reagents),a significantdrawbackofthismethodisthedetectedmutation spectrum,whichonlyincludesthosewhoseprobeshavebeen boundtothearrayandsubstitutionsordeletionsthatinclude onlyonenucleotide.42
Inthefollow-upstudyontheERICrecommendations,the AmpliChipdetected71%oftheTP53mutationsand81%ofthe patientswithmutations.Consideringthedetectionlimitand thetypesofmutationspotentiallydetectable,theAmpliChip identified91%ofthemutationsin91%ofthepatients.Fur- thermore,amongthe45mutationspresentinlessthan25%
ofthealleles,25(56%)weredetected.Thespectrumofmuta- tionsidentifiedbythismethodwasthesameasthatofthe DHPLCanalysisfollowedbytheSangersequencing.48
Dickeretal.34alsousedtheDHPLCfollowedbytheSanger sequencingandAmpliChipp53toscreenTP53mutationsin 193CLLpatients.BytheDHPLC/Sangersequencing,24ofthe 33(73%)mutationsweredetectedin20(77%)patients.Thus, theDHPLC/Sangersequencingdetected75%ofthemutations in80%ofthepatients.TheAmpliChipdetected30mutations (91%)in25patients(96%).TheAmpliChipdidnotdetectthree indels.Ontheother hand,it detectedeightmutationsthat werenotconfirmedbysequencing.Thediscrepancybetween thetwomethodologieswasaconsequenceofthelowsensi- tivityofthesequencing(10%ofthealleles),sincetheDHPLC initiallydetectedtheeightmutations.
Sangersequencing
FortheSangersequencing,itispreferabletousegenomicDNA than cDNA because somemutationsinthe TP53 may lead tomRNAdegradation(decayofnonsensemRNA).Exons4–10 mustbeanalyzed,astheyaccountformorethan95%ofthe mutations.42,45
Although it is a method available in most laborato- ries/centers and is considered the gold standard in the preciseidentificationofthe mutation,theSanger sequenc- ingisrelativelyexpensiveandtime-consuming.Furthermore, this method may fail to detect subclonal mutationswhen themutatedallele accountsforless than20–25%ofallthe alleles.42 These small mutated subclones are present in a significantpercentageofCLLpatientsandmightimplyaprog- nosisaspoorasthatassociatedwithmoreexpandedclones harboringTP53mutations.23,26,27
Next-generationsequencing(NGS)
Given its sensitivity and throughput, the targeted NGS is consideredthegold-standardmethodfordetectingsomatic mutationsintheTP53gene.23,25,26,35,49–51Italsotakeslesstime thanothermethodswhenitisnecessarytoanalyzealarge numberofsamplesormanyregionsofthegenome.Several commercial kits are currentlyavailable forthe preparation ofthesequencingreactionsofthe TP53and otherdisease- relatedgenes(suchasrelevantgenepanelsinoncology),either separatelyorinamultiplexformat.Dependingonthepur- pose,NGSplatformscanbeusedtoexaminealargenumber ofsamplessimultaneouslyoronlyafewsamplesforvarious genomicand/ordeepsequencingregions.42Thedeeporultra- deepsequencing allowsdetectingmutationswithverylow representability(subclones)atveryhighsensitivity.
Inthefollow-upstudyontheERICrecommendationspub- lished in 2012, samples from 69 patients were tested by ultra-deepNGS,withamediancoverageof27,538readsper base(range2096to88,976readsperbase).Theultra-deepNGS identifiedall 58 mutationsin theTP53, alsodetectedin37 patientsbyatleastoneoftheothermethods(FASAY,DHPLC andAmpliChip).Inaddition,othersubclonalmutationswere identifiedin24(65%)ofthesepatients.Among32patientsclas- sifiedasnon-mutatedbyother methods,the NGSdetected subclonalmutations(representedby0.2%–3.8%ofthealleles) ineight(25%)patients.Infiveofthem,theFASAYdetectedthe expansionofeachmutationinasubsequentsample.48Regard- ingthecorrelationbetweentheSangersequencingandNGS, anumberofstudiesshowedahighersensitivityofthelatter;
whileallvariantsdetectedbytheSangersequencingarealso detectablebytheNGS,theoppositeisnottrue.27,45,48,52,53
TheprimarychallengeinusingtheNGSisdataanalysis, particularlyinthedistinctionbetweensubclonalmutations and artifacts;thus, it isessential to have abioinformatics professional as a team member. To detect mutated alleles presentatlowlevels,thecoverage(numberofreadsperbase) mustbehighandtheerrorintroducedduringtheprocedure (polymeraseerrors,forexample)mustbelow.Thus,theuse of a polymerase with high fidelity (proofread)significantly improvesthedetectionlimitofthemethod.Statisticalanaly- sistoolsforresultsareunderdevelopment.42
The deep sequencing methodology was used in several recentstudiesthatdemonstratedtheimportanceofsubclonal mutationsin the TP53as apredictive factor inCLL. These studiesverifiedthattheresistancetotreatmentisassociated withtheexpansionofthesesubclones,alreadypresentatthe beginningoftreatment,butundetectablebyothermethods, andthattheacquisitionofnewabnormalitiesintheTP53in untreatedpatientsisarareevent.25,27,54
Inoneofthesestudies,theultra-deepNGSevaluated309 newlydiagnosedCLLpatientsanddetected85TP53mutations in46(15%)ofthem,includingmutationsaccountingforonly 0.3%ofthealleles.TheSangersequencingdidnotdetect50of the85mutations(59%)in15patients.Patientswithsubclonal mutationshadthesameclinicalphenotypeandsurvival(low) aspatientswithclonalmutations.Thestudyanalyzedsubse- quentsamplesandfoundthatthesmallsubclonesidentified beforethetreatmentbecamethepredominantcellpopulation inrelapseandanticipatedthedevelopmentofrefractoriness totherapy.27
In anotherstudy,the authorsre-analyzed samplesfrom 20patientsinitiallyclassifiedasnon-mutatedbytheFASAY and who had aTP53 mutationdetectedinrelapse, as well assamplesfrom40patientswhoremainedunchangedafter relapse. They usedthe deep NGS and ahigh-fidelity poly- merasetominimizesequencingerrors.Amongthe20relapsed patients,subclonal mutationsweredetectedintheTP53(in 0.20–3.71%ofthereads)in18(90%)ofthem.Amongthe40 patientswithoutrecurrence,onlyonepresentedamutation in 0.55%ofthe reads. Analyzingsubsequentsamples from this patient,itwas verifiedthattherewas noexpansionof theclone.25
OneimportantlimitationoftheNGSmethodisitshighcost andthefactthatthosepiecesofequipmentarenotavailable atmostmedicalcenters,particularlyinpoorcountries.
Othermethods
Oldermethodsbasedongelelectrophoresisarestillusedby somelaboratories.ExamplesofthesemethodsaretheSSCP (single strand conformation polymorphism) and the DGGE (denaturantgradient gelelectrophoresis), whichcan detect approximately5%ofthemutatedalleles.Theydonotdemand expensiveequipment,butarerelativelylaborious(especially thelatter)andneedextensiveoptimization.Also,theydonot identifytheprecisemutation,sotheyareconsideredasini- tialscreeningmethods(suchastheFASAYandDHPLC),with asubsequentneedforalteredsamples.18
Table1–Testsfordel(17p)andTP53mutationsinsomeofthemainlaboratoriesinBrazil.
Laboratory 1 2 3 4 5
Del(17p)
Method FISH FISHandMLPA FISHandMLPA FISHandMLPA FISHandMLPA
Deadline(days) 7 FISH10MLPANR FISH11MLPA28 FISH5MLPANR FISH15MLPANR TP53mutations
Method Sangersequencing Sangersequencing/NGS Sangersequencing Sangersequencing Sangersequencing
Limitofdetection NR 4%–15% NR NR 10%–12%
Deadline(days) 30 10 23 10 15
FISH,fluorescenceinsituhybridization;MLPA, multiplexligation-dependentprobeamplification;NR,notreported;NGS,next-generation sequencing.
Methodsfortheenrichmentofthemutatedallele
PurificationoflymphocytesorCD19+cellsfromtheperipheral bloodpromotestheenrichmentofthemutatedallelesinthe sampleandisusedinmostofthestudiesandtestsinCLL.
However,consideringtheneedtoidentifymutationsatvery lowsubclonallevels(<1%),greaterenrichmentwouldhaveto beperformedbeforemostofthemethodsmentionedabove, exceptthedeepNGS.TheCOLD-PCR(coagulationatalower denaturationtemperature-PCR)55andtheDISSECT(differen- tialstrandseparationatacriticaltemperature)56aremethods thatallowtheenrichmentofthemutationsbyapproximately 100–200times.Theformerisbasedonpreferentialdenatura- tionofthemutatedDNAduringPCRreactionsinwhichthe denaturationtemperature is reduced. Several denaturation temperaturesvaryingby0.3◦Careusedduringthereaction toallowenrichmentofdifferent typesofmutations.Thus, themutatedsequencesarepreferablyamplified,regardlessof theirlocationintheamplicon.55Astheinteractionbetween primerswithnon-specificproductformationisusuallyhigher intheCOLD-PCR,comparedtotheconventionalPCR,itisrec- ommendedtoconductthePCRusingwater-in-oilemulsion.57 The DISSECT is based on repeated cycles of hybridiza- tion and lower temperature denaturation between the targetedDNAandprobesrepresentingwildsegmentsofthe region/genetobeanalyzed.Theprobesareattachedtoasolid support(magnetic beads).Asthe denaturationtemperature isreduced,theDNAthatwilldissociatefromtheprobewill preferablybe themutated oneand it willbeeluted inthe supernatant.Mostofthewild-typeDNAwillremainbound tothebeads,whichareremovedfromthesolutionwiththe helpofamagnet.56
Itis important tonote that these methodsenrich only mutations that promote a reduction in the melting tem- perature of the amplicon. Furthermore, both require a pre-amplificationstepofthetotalgenome.55,56
TP53mutationdetectioninBrazil
Allfivelaboratoriesthatansweredthesurveyregardingthe TP53mutationdetectioninBrazilrelyontheSangersequenc- ing (4 laboratories) or the NGS and Sanger sequencing (1 laboratory)(Table1).Onlytwolaboratoriesreportedthelimit ofdetectionoftheirmethodsandtheonethatusestheNGS andSangersequencingreportedaslightlygreatersensitivity thantheonethatreliesonlyontheSanger.
Conclusion
InpatientswithCLLwhohaveundergoneatleastonetreat- mentline(i.e.,whorepresentrefractoryorrecurrentcases), the probability of the clonal expansion of cells with the TP53 mutationand/or17pdeletionisvery high.Thus,sev- eralmethodologieswillbeabletoidentifysuchchanges.To detect the 17pdeletion, the chromosome G-banding (kary- otype),FISHandarrays(SNP-arrayorCGH-array)ormoderate or high-coverage read NGS (about 100 or more reads per base)areconsideredthemosteffectiveones.Also,toidentify othersomaticmutationsinvolvingtheTP53gene,theSanger sequencing,precededornotbyascreeningmethod(ideally DHPLC)oramoderateorhigh-coveragereadNGS(about100 ormorereadsperbase),ismoreindicated.
Innewly-diagnosedCLLpatientsorinthosewhohavenot yet undergone treatment, the sensitivity of someof these methodsimpliesinfalse-negativeresultsincaseswithalow proportionofmutatedcells (<10–20% forsmallTP53 muta- tionsand<5%for17pdeletions).Forthesepatients,theideal methodwouldbetousethehigh-coverage/deepsequencing NGS(about1000ormorereadsperbase)andhigh-fidelitypoly- merase.Inthecaseofsmallmutations,theSangersequencing precededbyamethodofenrichmentofmutatedalleles,such astheCOLD-PCRorDISSECT,couldalsobesuitable(alsousing ahigh-fidelitypolymerase).
Foranyofthesemethods,itisessentialthatthelabora- toryperformanalyticalvalidation,documentingitsaccuracy, precisionandsensitivity/detectionlimit.InBrazil,thereare atleast fiveclinical laboratoriesthatoffer theFISH testto detect 17p deletions and the Sanger sequencing for TP53 mutations. The NGS is available in at least one of those labs.
Ofnote,thedetectionofthedel(17p)andTP53mutations playsacrucialrole,regardingthetreatmentdefinitionforCLL patients.Casesharboringthosealterationsdonotbenefitfrom theFCRschemeandotherchemoimmunotherapycombina- tionsthathaveresultedinthedramaticimprovementinthe responseandsurvivalratesofmostCLLpatientsinthelast decades.58–60Treatmentoptionsforpatientswiththedel(17p) and/or TP53 aberrant clones include ibrutinib, venetoclax, alemtuzumab,obinutuzumab,high-dosemethylprednisolone (HDMP)andrituximab,amongothers.Stemcelltransplanta- tioninalsoanoptioninhealthypatientswhoshowdisease progression.60,61
Financial support
ThisstudywassponsoredbyAbbvie.
Conflicts of interest
ThisstudywassponsoredbyAbbvie.
Acknowledgements
AbbVieparticipatedintheinterpretationofdata,reviewand approvalofthecontent.Alltheauthorshadaccesstoallrel- evantdataandparticipatedinwriting,reviewandapproval ofthismanuscript.EloisaMoreirafromKantarprovideddata collection,dataanalysis,literaturereviewandmedicalwrit- ingservicesinthedevelopmentofthismanuscript,fundedby AbbVie.
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