a universal method for elimination of hemolyzed plasma samples … · 2012. 9. 13. · a universal...
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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
Cancer
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A: PCA plot for microRNA signature performance
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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Sensitivity and linearity of Exiqons qPCR system
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Hemolysis have a strong effect on miR451 expression
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A: Effect of hemolysis on plasma miR-451 levels
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156 microRNAs
microRNAs affected by RBC contamination microRNAs not affected by RBC contamination
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microRNA expression profile from plasma samples with different levels of Red Blood Cell (RBC) contamination
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.
321 654
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PCA plot for microRNA profile among participating hospitals
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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)
Red Blood Cells• ~4.5 mL• ‘low’ RNA• ‘0’ DNA
Buffy coat• <0.1 mL• 10-100 µg RNA• 200-600 µg DNA
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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
Stage III
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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