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University of GroningenMass Spectrometry as a Highly Sensitive Method for Specific Circulating Tumor DNAAnalysis in NSCLCLamy, Pierre-Jean; van der Leest, Paul; Lozano, Nicolas; Becht, Catherine; Duboeuf,Frédérique; Groen, Harry J M; Hilgers, Werner; Pourel, Nicolas; Rifaela, Naomi; Schuuring,EdPublished in:CancersDOI:10.3390/cancers12103002IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2020Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Lamy, P-J., van der Leest, P., Lozano, N., Becht, C., Duboeuf, F., Groen, H. J. M., Hilgers, W., Pourel, N.,Rifaela, N., Schuuring, E., & Alix-Panabières, C. (2020). Mass Spectrometry as a Highly Sensitive Methodfor Specific Circulating Tumor DNA Analysis in NSCLC: A Comparison Study. Cancers, 12(10), 1-14.[3002]. her than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 22-04-2021

cancersArticleMass Spectrometry as a Highly Sensitive Method forSpecific Circulating Tumor DNA Analysis in NSCLC:A Comparison StudyPierre-Jean Lamy 1, *,† , Paul van der Leest 2,† , Nicolas Lozano 1 , Catherine Becht 3 ,Frédérique Duboeuf 3 , Harry J. M. Groen 4 , Werner Hilgers 5 , Nicolas Pourel 5 ,Naomi Rifaela 2 , Ed Schuuring 2,‡ and Catherine Alix-Panabières 6,‡123456*†‡Biopathologie et Génétique des Cancers, Institute d’Analyse Médicale Imagenome, Inovie, 6 Rue Fontenille,34000 Montpellier, France; nicolas.lozano@labosud.frDepartment of Pathology, University Medical Center Groningen, University of Groningen, Hanzeplein 1,9713 GZ Groningen, The Netherlands; p.van.der.leest@umcg.nl (P.v.d.L.); n.b.rifaela@umcg.nl (N.R.);e.schuuring@umcg.nl (E.S.)Oncologie Médicale, Clinique Clémenville, 25 rue Clémenville, 34000 Montpellier, France;catherine.becht@laposte.net (C.B.); frederique.duboeuf@gmail.com (F.D.)Department of Pulmonary Medicine, University Medical Center Groningen, University of Groningen,Hanzeplein 1, 9713 GZ Groningen, The Netherlands; h.j.m.groen@umcg.nlOncologie Médicale, Institute Sainte Catherine, 250 Chemin de Baigne Pieds, 84918 Avignon, France;w.hilgers@isc84.org (W.H.); n.pourel@isc84.org (N.P.)Laboratoire de Cellules Rares Circulantes, University Medical Center of Montpellier, 641, Avenue du DoyenGaston GIRAUD, 34093 Montpellier, France; c-panabieres@chu-montpellier.frCorrespondence: pierre-jean.lamy@labosud.fr; Tel.: 33-430-053-100Both authors contributed equally.Both senior authors contributed equally.Received: 23 September 2020; Accepted: 15 October 2020; Published: 16 October 2020 Simple Summary: We compared the UltraSEEK Lung Panel on the MassARRAY System(Agena Bioscience) with the FDA-approved Cobas EGFR Mutation Test v2 for the detectionof EGFR mutations in liquid biopsies of NSCLC patients, accompanied with preanalytical sampleassessment using the novel Liquid IQ Panel. For the detection of relevant predictive mutations usingthe UltraSEEK Lung Panel, an input of over 10 ng showed 100% concordance with Cobas EGFRMutation Test v2 and detection of all tissue confirmed mutations. In case of lower ccfDNA input,the risk of missing clinically relevant mutations should be considered. The use of a preanalyticalccfDNA quality control assay such as the Liquid IQ Panel is recommended to confidently interpretresults, avoiding bias induced by non-specific genomic DNA and low input of specific tumoralccfDNA fragments.Abstract: Plasma-based tumor mutational profiling is arising as a reliable approach to detect primaryand therapy-induced resistance mutations required for accurate treatment decision making. Here,we compared the FDA-approved Cobas EGFR Mutation Test v2 with the UltraSEEK Lung Panelon the MassARRAY System on detection of EGFR mutations, accompanied with preanalyticalsample assessment using the novel Liquid IQ Panel. 137 cancer patient-derived cell-free plasmasamples were analyzed with the Cobas and UltraSEEK tests. Liquid IQ analysis was initiallyvalidated (n 84) and used to determine ccfDNA input for all samples. Subsequently, Liquid IQ results were applied to harmonize ccfDNA input for the Cobas and UltraSEEK tests for 63 NSCLCpatients. The overall concordance between the Cobas and UltraSEEK tests was 86%. The Cobas test detected more EGFR exon19 deletions and L858R mutations, while the UltraSEEK test detectedmore T790M mutations. A 100% concordance in both the clinical (n 137) and harmonized(n 63) cohorts was observed when 10 ng of ccfDNA was used as determined by the Liquid IQ Cancers 2020, 12, 3002; ncers

Cancers 2020, 12, 30022 of 14Panel. The Cobas and UltraSEEK tests showed similar sensitivity in EGFR mutation detection,particularly when ccfDNA input was sufficient. It is recommended to preanalytically determine theccfDNA concentration accurately to ensure sufficient input for reliable interpretation and treatmentdecision making.Keywords: non-small cell lung cancer; EGFR mutation; circulating DNA; tyrosine kinase inhibitors;liquid biopsy1. IntroductionMutations and gene-fusions underlie key molecular mechanisms that drive cancer developmentand progression. Therefore, treatment strategies that target specific molecules related to gene mutationsor gene-fusions have been developed. In non-small cell lung cancer (NSCLC), activating mutationsof EGFR in exons 18–21 are well established as predictive biomarkers for treatment of patients withEGFR tyrosine kinase inhibitors (TKIs) [1]. Personalized treatment strategies for genetically stratifiedNSCLC subgroups improve patient outcomes, and molecular testing recommendations for treatmentwith targeted TKIs have been reported [2,3]. Despite the high response rates to various first- andsecond-generation EGFR-TKIs, eventually all patients with metastasized NSCLC with an EGFRmutation will develop disease progression due to acquired resistance, mostly attributable to the EGFRT790M mutation [4]. Osimertinib was introduced as a third-generation EGFR-TKI that selectively andirreversibly targets the EGFR T790M mutation [5]. In 2015, osimertinib was approved for treatment ofEGFR T790M-positive patients who have progressed on first- or second- generation EGFR-TKIs [6].In addition to EGFR T790M, other resistance mutations in EGFR emerged, e.g., C797S, L718Q, S768I andexon20 insertions [7]. Other mutated genes such as KRAS, BRAF, PIK3CA and ERBB2 have beenreported in patients treated with first- or second-generation and more recently third-generation EGFRinhibitors [4,8].EGFR mutation detection is nowadays performed with polymerase chain reaction (PCR)–basedmethods that are more sensitive than traditional Sanger sequencing and is currently implementedin many laboratories. As genetic mechanisms of secondary resistance arise in a sub-clonal manner,resulting in i.e., low-level allelic frequencies, secondary EGFR resistance mutation detection is evenmore challenging [9,10]. The development of resistance mutations and subsequent new treatmentoptions led to the necessity of evolutive molecular tumor profiling not only at time of diagnosis butalso at disease progression of patients receiving targeted therapy. However, of at least 20% of patients,clinicians are unable to obtain tumor tissue biopsy or evaluable tissue biopsy [11]. To solve this issue,liquid biopsy approaches have been introduced analyzing circulating tumor DNA (ctDNA) fromcell-free plasma. Several studies support the use of ctDNA to determine the EGFR mutational status incases where tumor tissue is limited or insufficient [12].In cancer patients, only a fraction of the circulating cell-free DNA (ccfDNA) carries tumor-specificsomatic mutations (i.e., the ctDNA fraction) against a background of circulating DNA originating fromperipheral blood cells and other tissues [13]. Thus, the ctDNA fraction might account for less than 1% inmany plasmas, and therefore it requires highly sensitive detection methods [13,14]. A major drawbackin the implementation of liquid biopsy approaches is that preanalytical and analytical factors have notbeen harmonized while their effects on performance and results of ctDNA assays are major [13–17].Variation of white blood cell (WBC)-derived DNA in cell-free plasma due to hemolysis during thecollection, transport and processing of blood is an essential factor that can affect PCR and sequencingresults by dilution of ctDNA with wild-type or non-cancerous DNA, increasing false-negative results,and erode reliable quantification of the variant allelic frequency (VAF).To date, only one of the very few FDA-approved ccfDNA-based tests with clinical utility is theCobas EGFR Mutation Test v2 (Roche Molecular Systems Inc., Pleasanton, CA, USA) detecting

Cancers 2020, 12, 30023 of 14mutations in ccfDNA from patients with lung cancer [5,18–22]. The Cobas test3 of14is approved as a companion diagnostic test on cell-free plasma to select NSCLC patients eligible forEGFR-TKI,includingosimertinibthe[23].In this study,we comparedvariant detection in 137 plasma samples of patients with NSCLCthisstudy, we comparedtheonvariantdetection in 137plasmasamplesof patientsNSCLCusingIntheUltraSEEK Lung Panelthe MassARRAYSystem(AgenaBioscience,SanwithDiego,CA, System (Agena Bioscience, San Diego, CA,usingtheUltraSEEK LungPanelontheMassARRAYUSA), a new highly-sensitive method to detect 67 mutations across 5 genes (EGFR, BRAF, KRAS, EGFRUSA), aandnewPIK3CA)highly-sensitivemethodto detect67 mutations5 genes(EGFR,BRAF,KRAS,ERBB2ERBB2in a one PCRmultiplexassay,with the acrossstandardmethodCobasMutation Panel Systemand PIK3CA)in a onePCR multiplexassay,the standardmethod CobasEGFR, MutationTest v2.Testv2. We alsointroducethe LiquidIQwithon the MassARRAYwhich provides System, which provides information onWe also introducethepreanalyticalLiquid IQ Panelon the MassARRAYinformationon theconditionsof each samplethrough the total input amount ofthe preanalyticalconditionsof eachthroughthe total orinputamountof amplifiableamplifiableccfDNAin a reaction,thesampleccfDNAsize (presenceabsenceof evelDNA) and the level of WBC contamination, and it authenticates samples using a comprehensivesetof WBCand it authenticatessamplesusingstudieda comprehensiveset leotide polymorphisms(SNPs).We thenthe optimalccfDNA andpolymorphismsWeIQthenstudiedthe optimalof ccfDNA determined with the Liquiddeterminedwith (SNPs).the Liquidits implicationon inputtests results.IQ and its implication on tests results.2. Results2. Results2.1. Comparison of Mutation Detection Using UltraSEEK and Cobas 2.1. Comparison of Mutation Detection Using UltraSEEK and Cobas Plasma-derived ccfDNA of 137 patients was tested for mutation-harboring ctDNA using thePlasma-derivedccfDNApatients wastested formutation-harboringthe SystemUltraSEEK Lung Panelon ofthe137MassARRAYandcompared to the ctDNAresults usingfrom the UltraSEEK Lung Panelthe MassARRAYSystemandwithcomparedto thethe diagnostic resultsdiagnosticreportsof theonsameplasma samplestestedthe CobasEGFR fromMutationTest v2, EGFR Mutation Test v2, according ccording to the conditions as recommended by the manufacturers. For UltraSEEK analysis, thethe conditionsas recommendedthe manufacturers.analysis,numbertheofnumberof amplifiablecopies as bydeterminedby Liquid ForIQ UltraSEEK analysis wereused tothecalculate analysis were used to calculate the ccfDNA inputamplifiablecopiesasdeterminedbyLiquidIQccfDNA input for each sample. Consequently, the ccfDNA input for the UltraSEEK and Cobas analyses werefor each sample.the ccfDNAinputfor theUltraSEEK andCobaswasanalyseswere notConsequently,equal. Concordantdetectionof EGFRmutationsby bothpanelsfound in thpanelswasfoundin118cases (86%; cases (86%; Figure 1A). In 10 cases (7.3%) the Cobas detected EGFR mutations missedwith detected EGFR mutations missed with UltraSEEK , while theFigure1A).In10cases(7.3%)theCobas UltraSEEK , while the UltraSEEK detected mutations in 9 cases missed by Cobas (6.6%; Table (6.6%; Table S1; Table S2). Cohen’sUltraSEEK detectedκmutations9 cases missedby CobasS1;Table S2). Cohen’srevealed a insubstantialagreementof 0.71 (TableS3). Considering all bleS3).Consideringallclinicallyrelevantrelevant mutations covered by the UltraSEEK Panel (Table S1), heUltraSEEK .2%of allwithpatients in 18.2% of all patients not detectable with the Cobas test, compared to 8.0% detectedthe not detectablewiththe Cobastest,to 8.0%withthe Cobastest onlyand(Figure1B; test only Cobas(Figure1B; TableS3).comparedAccordingly,the detectedagreementbetweenUltraSEEK Cobas drops to a moderate level K andCobasdrops to a moderate level of 0.54 (Table S2).0.54 (Table S2).42 EGFRCancers2020,hotspot12, xFigure 1. Comparison of mutation detection between UltraSEEK and Cobas . Pie charts representing . Piethe sampleswhich wereofconcordantand Cobas and(blue),samplesin chartswhichFigure1. Comparisonmutation betweendetectionUltraSEEK between UltraSEEK Cobas detected UltraSEEK smoremutationsrepresenting the samples which were concordant between UltraSEEK and Cobas (blue), samplesin which UltraSEEK detected more mutations (green), and samples in which Cobas detected moremutations (red) for (A) the mutations detectable on both panels and (B) all clinically relevantmutations across BRAF, EGFR, KRAS, ERBB2 and PIK3CA. (C) Bar graph illustrating the total amountof mutations detected in all 137 samples. The blue bar represents the number of mutations that could

Cancers 2020, 12, 30024 of 14(red) for (A) the mutations detectable on both panels and (B) all clinically relevant mutations acrossBRAF, EGFR, KRAS, ERBB2 and PIK3CA. (C) Bar graph illustrating the total amount of mutationsdetected in all 137 samples. The blue bar represents the number of mutations that could be detected onboth panels;Cancers2020, 12, thex green bar represents detected mutations unique for the UltraSEEK Panel. One EGFR4 of 14exon19 deletion genotype was detected with Cobas that is not represented on the UltraSEEK Panel(redbebar).(D) Barillustratingfrequencyof detectionper mutationUltraSEEK (green) anddetectedon graphboth panels;the greenbar representsdetectedmutationswithuniquefor the UltraSEEK Cobas(red).Panel.One EGFR exon19 deletion genotype was detected with Cobas that is not represented on theUltraSEEK Panel (red bar). (D) Bar graph illustrating frequency of detection per mutation withA totalof 115 EGFRcovered by both tests were detected with both the UltraSEEK UltraSEEK (green)mutationsand Cobas (red).and Cobas panels (Table S1; Figure 1C). Using UltraSEEK , more EGFR exon19 deletions andA total of were115 EGFRmutationscoveredboth testswere detectedwith bothOnthetheUltraSEEK L858R mutationsmissedcomparedwithbyCobas(9 versus2, respectively).other hand, missedCobaspanelsS1; Figure1C).mutationUsing UltraSEEK ,deletionsandthe andCobasthe (TableTKI-resistantT790Min 6 out of 34morecases,EGFRwhileexon19UltraSEEK detected (9 versus 2, respectively). On the other hand,L858RmutationsweremissedcomparedwithCobasall (Figure 1D; Table S2). missed the TKI-resistant T790M mutation in 6 out of 34 cases, while UltraSEEK detectedtheCobasTheUltraSEEK analysis revealed 20 additional mutations, partially other than EGFR mutations,all(Figure1D;TableS2). by the Cobas Panel (Figure 1C; Table S1). Among these mutations wereand those were not coveredUltraSEEK analysis revealed 20 additional mutations, partially other than EGFRcommonTheKRASG12/13 mutations, as well as EGFR C797S and BRAF V600E mutations frequentlymutations, and those were not covered by the Cobas Panel (Figure 1C; Table S1). Among theseassociated with treatment resistance (Figure 1D; Table S1).mutations were common KRAS G12/13 mutations, as well as EGFR C797S and BRAF V600E mutationswith 2.2. frequentlyValidation associatedof the LiquidIQ treatmentPanel resistance (Figure 1D; Table S1). Liquid Panel2.2.ValidationtheTheLiquid ofIQPanelIQonthe MassARRAY System is a novel approach to quantify andqualify theextracted ccfDNA prior to UltraSEEK analyses. In order to validate its applicability,The Liquid IQ Panel on the MassARRAY System is a novel approach to quantify and qualify84 randomly-selectedpatient-derivedccfDNA samplesextractedfromtoplasmacollectedfrom the samethe extracted ccfDNAprior to UltraSEEK analyses.In ordervalidateits k(n 42)orEDTA(n ed patient-derived ccfDNA samples extracted from plasma collected from thesameusingthe LiquidIQ Panel.numbersof amplifiableand lyin Similareither Streck(n 42)or EDTA (n copies 42) bloodcollectionwere analyzed Qubit measureshigherconcentrationsusing the Liquid IQ Panel. Similar numbers of amplifiable copies and yield were determined and Qubit was found (R2 0.87;of ccfDNAS1B),EDTAa strongcorrelationbetween(FigureStreck andsamples(Tablebetween1; FigureLiquidS1A). IQDespitethat Qubit measures higherFigure2).concentrationsof ccfDNA (Figure S1B), a strong correlation between Liquid IQ and Qubit wasfound (R2 0.87; Figure 2).Table 1. Liquid IQ results for the Streck and EDTA samples.Table 1. Liquid IQ results for the Streck and EDTA samples.Tube TypeSNP CallsTube TypeSNP CallsStreck (n 42) 20 (16–21)Streck (n 42)20 (16–21)EDTA (n 42) 20 (15–21)AmplifiableWBCYield (ng/µL)Qubit (ng/µL)AmplifiableWBC Contamination Yield (ng/µL) Qubit (ng/µL)CopiesCopies Contamination179 (57–734)3% (0–28%)0.40 (0.13–1.6) 0.62 (0.43–3.3)179 (57–734)3% (0–28%)0.40 (0.13–1.6)0.62 (0.43–3.3)177 (66–1010)1% (0–35%)0.39 (0.15–2.3) 0.63 (0.49–4.2)No Result *No Result *000EDTA (n 42)20 (15–21)177 (66–1010)1% (0–35%)0.39 (0.15–2.3)0.63 (0.49–4.2)0Data are presented as median with range between brackets. * Level of amplifiable copies too low forData are presented as median with range between brackets. * Level of amplifiable copies too low for accurate callingcallingand considered unevaluable.and accurateconsideredunevaluable. andFigure2. CorrelationbetweenLiquidIQIQand Qubit Qubit resultsresults of StreckFigure2. CorrelationbetweenLiquidStreck andandEDTAEDTAplasmaplasmasamples.samples. 2 correlationbetweenLiquidQubit for ccfDNAthe LiquidIQ IQandandQubit for theextractedfromfromEDTA(black;R2 R0.91)22andStreckR separately 0.82) separatelyshowedresults.and0.91)Streck(red;R (red; 0.82)showedsimilarsimilarresults.2.3. Quantitative and Qualitative Analysis Using the Liquid IQ PanelAll 137 clinical plasma samples were analyzed with the Liquid IQ Panel to calcula

Cobasfi EGFR Mutation Test v2 (Roche Molecular Systems Inc., Pleasanton, CA, USA) detecting Cancers 2020 , 12, 3002 3 of 14 42 EGFR hotspot mutations in ccfDNA from patients with lung cancer [5,18–22].

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