A universal method for elimination of hemolyzed plasma samples … · 2012. 9. 13. · A universal...

A universal method for elimination of hemolyzed plasma samples that improves miRNA signature performance for early detection of colorectal cancerPeter Mouritzen1, Søren J. Nielsen1, Nana Jacobsen1, Jan Stenvang2, Thorarinn Blondal1, Torben Ørntoft3, Nils Brünner2, Claus L. Andersen3, Hans J. Nielsen4 and Adam Baker1 1Exiqon A/S, Vedbaek, Denmark; 2University of Copenhagen, Copenhagen, Denmark; 3Aarhus University Hospital, Aarhus, Denmark; 4Hvidovre Hospital, Copenhagen, Denmark

Conclusions and future prospects•Aplasma/serummicroRNAPCRprofilingplatformwithunparalleledsensitivitywasdeveloped•Asimpleworkflowfromsamplepreparationtodataanalysisallowstestresultsfrom100uLof plasmawithinoneworkingday•HemolysiswasdemonstratedtobeamajordeterminantofsamplequalityformicroRNAprofiling•HemolysiscorrelatedwithhospitalIDandwiththeuseofspecificplasmacollectiontubes•Analysisofthrombocytecontaminationison-going•AplasmamicroRNAsignaturebasedonalimitednumberofmicroRNAswasdevelopedinone hospital and validated in samples from independent hospitals•Validationofthesignatureinalargersetof>1000samplesfrommultiplehospitalsison-going•Aprospectivetrialcollecting5000samplesforvalidationhasbeeninitiated

ThisworkwassupportedbytheDanishAdvancedTechnologyFoundationgrant007-2009-2

ConcerningmiRCURYLNA™UniversalRTmicroRNAPCR:NOTICETOPURCHASER:LIMITEDLICENSEPurchaseofthisproductincludesanimmunityfromsuitunderpatentsspecifiedintheproductinserttouseonlytheamountpurchasedforthepurchaser’sowninternalresearch.Nootherpatentrightsareconveyedexpressly,byimplication,orbyestoppel.FurtherinformationonpurchasinglicensesmaybeobtainedbycontactingtheDirectorofLicensing,AppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,USA.

Figure 5. Effect of hemolysis on plasma microRNA profiles. TheRBCdilutionserieswasprofiledfortheexpressionof378microRNAs.ThemicroRNAsweresortedbasedonmaximalchangeofexpressionaftertheadditionofRBClysate.microRNAsaffectedbyandimperviousto hemolysis are indicated.

Figure 9. Biomarker discovery and classifier selection. 100samplesfromhospital2wereusedtogeneratealistofcandidatemarkers.The64mostdifferentiallyexpressedmicroRNAswereusedtogenerateaPCAplot(A).AsignaturebasedonalimitedsetofmicroRNAswasdevelopedandevaluatedusingreceiver-operatorcharacteristicscurve(B).

Table 4. Performance of the microRNA signature in the different sample sets.

All samples Hospital 2(discovery)

Remaining hospitals(validation)

AUC 0.68 0.81 0.87

Sensitivity(%) 67 75 82

Specificity(%) 65 79 89

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B: ROC curve for microRNA CRC signature

Figure 3. Superior sensitivity and linearity of the LNA™-enhanced miRNA RT-qPCR Platform. Apoolofsynthetictemplatesfor647microRNAswassubjectedtoserialdilution(thelowestinputrepresents15copiesofeachtemplateRNAinthePCRreaction)andthenassayedbyRT-qPCR.ThemedianCtvalueforallassayswasthenplottedagainsttemplateconcentration,demonstratinglinearityoftheassayplatformdownto15copiesoftemplate.

Figure 4. Effect of hemolysis on plasma miR-451 levels. Toppanel:Anon-hemolyzedplasmasamplewasspikedwithincreasingamountsofredbloodcell(RBC)lysateandanalyzedforhaemoglobincontent(A414)andmiR-451levels(qPCR).Bottompanel:Aselectionof50plasmasamplesfromthestudywasanalyzedforhaemoglobincontentandmiR-451levels.Potentialcut-offvaluesare indicated by red lines.

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Table 3. Use of plasma collection tubes at participating hospitals. 1)GreinerVacuette,2)BDVacutainer,3)TerumoVenosafe.Hospitalswithhighincidenceofhemolysisarehighlightedinred.

Discovery and validation of a plasma-based microRNA signature for early detection of CRCAsetof325plasmasamplesfromCRCpatientsandhealthycontrolsfrom7Danishhospitals(Table2)wasprofiledacross378microRNAscommonlyfoundinhumanplasma.Initialanalysisofthedataset demonstratedpoorseparationbetweenthegroups(ROCAUC=0.68,Table4).Principalcomponentanalysis(PCA)ofallcontrolsamplesshowedmarkeddifferencesinmicroRNAprofilesbetweenthedifferenthospitals,suggestingthatthesedifferencesmayhampertheidentificationofgoodCRC-relatedbiomarkers.Wethereforeevaluatedasetofparametersrelatedtosampleacquisitionandstorage.Onesuchparameter,hemolysis(orthepresenceofredbloodcelllysateinthesample),wasshowntodiffergreatlybetweenhospitals,andtocorrelatewiththeoutlierhospitalsinourPCA(Figures7-8).

Wetheneliminatedsamplesfromtheoutlierhospitalsandusedthedatafromhospital2(N=100)todevelopasignatureofalimitednumberofmicroRNAsthatdistiguishwellbetweenCRCandcontrols(AUC=0.81,Figure9andTable4).Finally,wewereabletovalidatethesignatureinsamplesfromtheotherhospitalsnotaffectedbyhemolysis(AUC=0.87,Table4).

Table 2. Clinical material for signature discovery and validation. Plasmasampleswereassayedforthepresenceof378microRNAsbyRT-qPCR.

Cancers Controls

Mean age(Range) 70(40-94) 69(33-93)

GenderMale 86 79

Female 83 77

StageII 119 N/A

III 50 N/A

Figure 7. Variation in microRNA profile among participating hospitals. Theprofilesofthe131microRNAsexpressedinall153controlsampleswereusedasinputinaPCA. ThefirstandsecondcomponentsandthehospitalIDsareindicatedonthefigure.

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Figure 8. Variation in selected microRNAs among participating hospitals. TheprofilesofmiR-122(un-affectedbyhemolysis)andmiR-451(hemolysismarker)areshown.Hospitals1,5,6allcontributedamajorproportionofhemolyzedsamples.

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Serum/Plasma microRNAs are promising disease biomarkers• Playimportantregulatoryroleinmanydiseases including cancers• Integratesbiologyfromentireorganismincluding diseased tissue• Minimallyinvasive• Routinelyobtainedinhospitalsandatgeneralpractitioners• Largehistoricalcollectionsexistfordiscovery• microRNAsarepresentinlowbutdetectableamounts• PlasmamicroRNAsarestableunderstandardsampling and storage conditions

Three challenges of working with serum/plasma• Serum/plasmacontainsRNasesandEnzymeinhibitors• Serum/plasmacontainslowamountsofnucleicacid• Serum/plasmaiscell-freebutmaybecontaminatedbybloodcells

Our solutions• Avoidtheuseofheparincollectiontubes• Usespike-instomonitorforco-purificationofinhibitors/RNases• UsecarrierRNAduringpurification• UseasensitiveanalyticalsystemsuchastheLNA™-enhancedmiRNA RT-qPCRplatform• Avoidtransferofcellularmaterialduringsampleacquisition• Minimizehemolysisduringacquisition• ProcessplasmaatRTwithin2hrsofphlebotomy• QCplasmaforhaemoglobincontent• MonitorPCRdatasetforsignsofhemolysis

Figure 1. Clinical Source of biomarker – 10 mL blood collection. Nucleicacidspresentindifferentbloodfractionsfroma10mlbloodsample.

Figure 2. Effect of carrier RNA.Plasma(200µL)fromtwoindividualswaspurifiedintheabsenceorpresenceofcarrierRNA(MS2phageRNA)andassayedbyRT-qPCRforthepresenceofthreemiRNAs.NotetheincreaseddetectionanddecreasedvariabilityinsamplespurifiedwithcarrierRNA.

10 mL blood sample

Blood plasma contains small amount of RNA

Plasma• ~5.5 mL• 1-50 ng RNA• <100 ng DNA ( in disease)

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Colorectal cancerColorectalcancer(CRC)isamajorcauseofmortalityinthewesternworld.EarlydetectionofCRCimprovessurvivalandscreeningforCRChasbeenclinicallyproventolowerCRC-relatedmortality.However,although population screening programs have been implemented in a number of countries, screening ratesamongthe50-75yearoldsareunsatisfactory.

There is therefore a clear unmet need for a quick, sensitive, specific, and minimally invasive screening assay to select at risk individuals for definitive diagnosis by colonoscopy.

Figure 6. Colorectal cancer stages.

Stage IStage 0

Stage II

Spread to other organs

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Stage IV

Table 1. Colorectal cancer survival rates in US. EarlydetectionofCRCimprovessurvival.

CRC Stage 5 year relative survival Treatment

0-I 93% Surgery

II 80% Surgery

III 58% Surgery/adjuvantchemotherapy

IV 6.9% Chemotherapy

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