Circulating mutational portrait of cancer : manifestation of aggressive clonal events in both early and late stages

RESEARCH Open Access Ci cula ing mu a ional po ai o cance : mani es a ion o agg essi e clonal e en s in bo h ea ly and la e s ages Meng Yang 1,2,11,12 , Umi Topaloglu 1,2,12 , W. Je ey Pe y 1,3,12 , Ma hew Pagni 1,7,12 , K is ie L. Foley 1,9,12 , S e an C. G an 1,3,12 , Mac Robinson 1,12 , Rhonda L. Bi ing 1,3,12 , Alexand a Thomas 1,3,12 , Angela T. Alis a 1,3,12 , Rodwige J. Desnoye s 1,3,12 , Michael Goodman 1,3,12 , Ca ol Alb igh 1,3,12 , Me cedes Po osnicu 1,3,12 , Mihaela Va ca 1,3,12 , Shadi A. Qasem 1,5,12 , Ba y DeYoung 1,5,12 , Ville Ky ola 1,2,10,12 , Ma i Nyk e 10 , Kexin Chen 11 , Edwa d A. Le ine 1,6,12 , Edga D. S a en 1,6,12 , Ralph B. D’Agos ino J 1,4,12 , Robin M. Pe o 1,3,12 , William Blacks ock 1,8,12 , Baya d L. Powell 1,3,12 , Edwa d Ab aham 1,2,12 , Bo is Pasche 1,2,3,12* and Wei Zhang 1,2,12,13,14* Abs ac Backg ound: Solid umo s esiding in issues and o gans lea e oo p in s in ci cula ion h ough ci cula ing umo cells (CTCs) and ci cula ing umo DNAs (c DNA). Cha ac e iza ion o he c DNA po ai s and compa ison wi h umo DNA mu a ional po ai s may e eal clinically ac ionable in o ma ion on solid umo s ha is adi ionally achie ed h ough mo e in asi e app oaches. Me hods: We isola ed c DNAs om plasma o pa ien s o 103 lung cance and 74 o he solid umo s o di e en issue o igins. Deep sequencing using he Gua dan 360 es was pe o med o iden i y mu a ions in 73 clinically ac ionable genes, and he esul s we e associa ed wi h clinical cha ac e is ics o he pa ien . The mu a ion p o iles o 37 lung cance cases wi h pai ed c DNA and umo genomic DNA sequencing we e used o e alua e clonal ep esen a ion o umo in ci cula ion. Fi e lung cance cases wi h longi udinal c DNA sampling we e moni o ed o cance p og ession o esponse o ea men s. Resul s: Mu a ions in TP53,EGFR, and KRAS genes a e mos p e alen in ou coho . Mu a ion a es o c DNA a e simila in ea ly (I and II) and la e s age (III and IV) cance s. Mu a ion in DNA epai genes BRCA1,BRCA2, and ATM a e ound in 18.1% (32/177) o cases. Pa ien s wi h highe mu a ion a es had signi ican ly highe mo ali y a es. Lung cance o ne e smoke s exhibi ed signi ican ly highe c DNA mu a ion a es as well as highe EGFR and ERBB2 mu a ions han e e smoke s. Compa a i e analysis o c DNA and umo DNA mu a ion da a om he same pa ien s showed ha key d i e mu a ions could be de ec ed in plasma e en when hey we e p esen a a mino clonal popula ion in he umo . Mu a ions o key genes ound in he umo issue could emain in ci cula ion e en a e on line adio he apy and chemo he apy sugges ing hese mu a ions ep esen ed esis ance mechanisms. Longi udinal sampling o i e lung cance cases showed dis inc changes in c DNA mu a ion po ai s ha a e consis en wi h cance p og ession o esponse o EGFR d ug ea men . (Con inued on nex page) * Co espondence: [email p o ec ed];wezhang@wakeheal h.edu 1 Wake Fo es Bap is Comp ehensi e Cance Cen e , Wake Fo es Bap is Medical Cen e , Medical Cen e Bl d., Wins on-Salem, NC 27157, USA Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2017 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 DOI 10.1186/s13045-017-0468-1 (Con inued om p e ious page) Conclusions: This s udy demons a es ha c DNA mu a ion a es in he key umo -associa ed genes a e clinical pa ame e s ele an o smoking s a us and mo ali y. Mu a ions in c DNA may se e as an ea ly de ec ion ool o cance . This s udy quan i a i ely con i ms he hypo hesis ha c DNAs in ci cula ion is he esul o dissemina ion o agg essi e umo clones and su i al o esis an clones. This s udy suppo s he use o c DNA p o iling as a less-in asi e app oach o moni o cance p og ession and selec ion o app op ia e d ugs du ing cance e olu ion. Keywo ds: Liquid biopsy, Non-in asi e, Clonali y, Mu a ion a e, Lung cance Backg ound Tumo s ha e been b oadly classi ied as ei he hema opoie ic o solid ypes. Hema opoie ic cance s a e de i ed om neoplas ic cells o blood cell o igin, and solid umo s a e no mally associa ed wi h a speci ic o gan o issue ype. In he molecula and genomic e a o medicine, he e a e wo seemingly opposi e ajec o ies in he cha ac e iza ion o cance . One ajec o y is he zoom- in app oach, which di ides each cance ype u he in o sub ypes. In his e o , a adi ionally ecognized cance ype, such as lung cance , can be u he di ided in o mul iple sub ypes based on he molecula and genomic signa u e [1]. The opposing ajec o y is he me ging o mul iple cance ypes in he adi ional a ena in o a com- mon “molecula ”cance ype. F om his pe spec i e, o a - ian and p os a e cance cases can be mo e simila o each o he a he han cases wi hin hei own o gan-speci ic can- ce s [2–4]. The bounda ies a e also blu ed be ween blood cance s and solid umo s wi h he ecogni ion o CTCs in he blood o solid umo cance pa ien s. CTCs ha e been emphasized as he ansi o y cells o a solid umo lea ing one o gan and mo ing o ano he ia he blood o lymph- a ic sys em esul ing in me as a ic disease. Ad ance- men in de ec ion echnologies has inc easingly shed ligh on he impo ance o CTCs in cance diagnosis and p ognosis [5–12]. I has long been ecognized ha umo cells can elease p o ein molecules in o ci cula ion, e.g., umo ma ke s such as PSA, CEA, and CA125 [13–15]. This, howe e , can occu wi hou umo cells en e ing ci cula ion [16, 17]. The ecogni ion ha umo cells do en e ci cula ion led o he logical conclusion ha all molecula e en s occu ing in solid umo cells migh be e lec ed in ci cula ion [18–22]. Indeed, mu a ions o umo - ela ed genes and epigene ic al- e a ions in DNA me hyla ion de ec ed in umo issues ha e been de ec ed in CTCs and cell- ee plasma o he pa- ien s o all solid cance ypes examined [23–25]. Wi h he capaci y o nex gene a ion sequencing signi ican ly inc eas- ing in ecen yea s, blood based “liquid biopsy”has gained ecogni ion as a non-in asi e app oach o gain insigh in o he molecula and genomic-d i e e en s du ing solid umo p og ession, as a window o moni o cance esponse o ea men , and as a means o guide a ge ed he apies based on de ec ed ac ionable mu a ions [26–30]. The e a e, howe e , many unanswe ed ques ions e- ga ding his eme ging app oach. Fi s , will mu a ion a es de ec ed in c DNA be as aluable as mu a ion p o iling in umo issues? Second, is he e a speci ic mechanism o dissemina e umo issue mu a ions in o ci cula ion? Thi d, would hese me as asis- ela ed e en s se e as mo e e ec i e a ge s o in e en ion? In his s udy, we e alua ed he clinical alue o assessing c DNA using he Gua dan 360 pla o m in 177 pa ien s wi h solid umo s, he majo i y o whom we e ea ed o lung cance . The esul s allow us o e alua e mu a ional po ai s o solid umo s in ci cula ion. We also p esen he ini ial success in applica ion o his new genomics pla - o m in cance moni o ing and ea men . Me hods Pa ien s and sample collec ion We e ospec i ely e iewed he liquid biopsy esul s o 177 consecu i e de-iden i ied pa ien s wi h di e se cance s who we e seen a Wake Fo es Bap is Comp ehensi e Cance Cen e . Blood samples we e collec ed be ween June 2015 and Sep embe 2016. Sociodemog aphic and clinical da a including gende (male, emale), age (<55, 55–65; 65.1–75; >75), body mass index (unde weigh , no - mal, o e weigh , obese), smoking his o y (cu en / ecen , o me , ne e ), ace (whi e, black, Asian, o he ), s age (I, II, III, IV, unknown), me as asis (0, 1, 2, 3+), umo ype, and i al s a us (ali e, deceased) o each pa ien we e col- lec ed. Smoking s a us was de ined by sel - epo ed smok- ing his o y ob ained om Cance Regis y and/o Epic Elec onic Medical Reco d. Ne e smoke s we e de ined as esponden s who smoked less han 100 ciga e es in hei li e ime. Based on e idence ha smoking cessa ion educes cance isk by hal a 5 yea s, ac i e smoke s a he ime o clinical da a collec ion and hose who had qui smoking wi hin he p e ious 5 yea s we e conside ed cu en / ecen smoke s [31]. We de ined lung cance s as lung adenoca cinoma (LUAD), lung squamous ca cinoma (LUSC), non-small cell lung cance -no o he wise speci- ied (NSCLC-NOS), and small cell lung cance (SCLC), acco ding o WHO classi ica ion. O e all su i al was de- ined as he in e al om he da e o ini ial su gical esec- ion o he da e o las ollow-up o dea h. Mos o he s a is ical analysis was desc ip i e in na u e. The sample Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 Page 2 o 13 size was de e mined by he a ailable pa ien s wi h gene ic es ing in o ma ion. We also examined 37 lung cance is- sue samples wi h comple ed sequencing ha we e pai ed wi h blood samples. The s udy was app o ed by he local ins i u ional e iew boa d, and all pa ien s p o ided w i - en in o med consen o gene ic analysis o hei umo and plasma samples p io o pa icipa ion in his s udy. Ex ac ion and quan i ica ion o c DNA and DNA Blood samples (3–10 ml) we e collec ed in EDTA ubes (BD Vacu aine , Beck on, Dickinson and Company) and cen i uged o 10 min a 1000 gwi hin 4 h o he blood d aw. The supe na an con aining he plasma was u he cen i uged a 14,000 g o 10 min a oom empe a u e and was s o ed a −80 °C un il analysis. Ini ially, he plasma samples we e selec ed on he basis o hei a ail- abili y, and hen, consecu i ely. DNA was ex ac ed om plasma using he QIAamp DSP Vi us Ki (Qiagen), ac- co ding o he manu ac u e ’s ins uc ions. A eal- ime quan i a i e PCR TaqMan Assay a ge ing GAPDH was used o measu e plasma DNA concen a ion. DNA was ex ac ed om he esh ozen biopsy sample using he AllP ep DNA/RNA Mini Ki (Qiagen) and was quan i ied wi h Qubi 2.0 (Li e Technologies). Iden i ica ion o genomic mu a ions by NGS Nex gene a ion digi al sequencing was pe o med using he Gua dan 360 es by Gua dan Heal h., (Redwood Ci y, CA; www.gua dan heal h.com), a Clinical Labo a o y Im- p o emen Amendmen s (CLIA)-ce i ied and College o Ame ican Pa hologis s (CAP)-acc edi ed clinical labo a o y (Gua dan Heal h, Inc.). A he ime o his s udy, his es iden i ies po en ial umo - ela ed genomic al e a ions ia comple e exon sequencing o 73 cance - ela ed genes in c DNA ex ac ed om plasma. c DNA was ex ac ed om plasma, and he amoun o c DNA was quan i ied using elec opho e ic sepa a ion in a massi ely pa allel capilla y a ay sys em allowing o pos -ex ac ion high- h oughpu , high- esolu ion agmen size-speci ic da a ac- quisi ion o each sample p ocessed. The c DNA was hen analyzed by pai ed-end sequencing by syn hesis u ilizing an Illumina Hi-Seq 2000 pla o m and hg19 as he e e ence genome as desc ibed [32]. Digi al sequences we e econ- s uc ed using Gua dan Heal h’s p op ie a y bioin o ma ics algo i hms, allowing he de ec ion o 1–2mu an agmen s in 10 mL o blood wi h an analy ic speci ici y g ea e han 99.9999%. Single nucleo ide a ian s (SNV), a ian s o unce ain signi icance (VUS), ampli ica ion, dele ion, and usions we e quan i a i ely epo ed. Thi y-se en lung issue samples we e p ocessed by Founda ion Medicine (Bos on, MA) using he Founda- ionOne NGS panel. Fo he Founda ionOne es , DNA was ex ac ed om one o mo e 40-μmsec ionso FFPE issue using he Maxwell 16 FFPE Plus LEV DNA Pu i ica ion ki (P omega) and was quan i ied using a s anda dized PicoG een luo escence assay (In i ogen). Lib a y cons uc ion was pe o med using 50–200 ng o DNA shea ed by sonica ion o app oxima ely 100–400 bp be o e end- epai , dA addi ion and liga ion o indexed, Illu- mina sequencing adap o s. En ichmen o a ge sequences was achie ed by solu ion-based hyb id cap u e wi h cus om bio inyla ed oligonucleo ide bases. En iched lib a ies we e sequenced o an a e age median dep h o >500× wi h 99% o bases co e ed >100× (IlluminaHiSeq 2000 pla o m using 49 × 49 pai ed-end eads) and we e mapped o he e e ence human genome (hg19) using he Bu ows- Wheele Aligne and he publicly a ailable SAM ools, Pica d, and Genome Analysis Toolki . Genomic al e a ions de ec ed include base subs i u ions, inse ions, dele ions, copy numbe al e a ions, and selec ed gene usions (h p:// ounda ionone.com/). Poin mu a ions we e iden i ied by a Bayesian algo i hm; sho inse ions and dele ions de e - mined by local assembly; gene copy numbe al e a ions iden i ied by compa ison o p ocess-ma ched no mal con ols; and gene usions/ ea angemen s de e mined by clus e ing chime ic eads mapped o a ge edin ons[33]. S a is ical analysis Demog aphic in o ma ion such as gende , age, BMI, smok- ing his o y, ace, s age, me as asis, i al s a us, umo ype, as well as he da es o sample ecep ion, da es o esul s, lis o al e a ions, and d ug(s) a ailable we e ex ac ed om he medical epo s and we e analyzed. Non-synonymous som- a ic mu a ion calls we e quan i ied. Pa ien s we e assigned o low o high mu a ion load g oups based on he coho median mu a ion numbe . We applied a Mann-Whi ney- Wilcoxon ank sum [34] and a K uskal-Wallis es [35] o compa e mu a ion load wi h clinical a iables wi h wo g oups and mul iple g oups, espec i ely. A Fishe ’sexac es was used o de e mine associa ion be ween smoking s a us (de ined as an o dinal a iable—cu en / ecen , o me , ne e smoke s) and DNA damage genes. O e all su i al was es ima ed using he Kaplan-Meie me hod. We e alua ed he o e all su i al using he log- ank es . All signi icance es s we e wo-sided. All lung cance pa ien s wi h a ailable c DNA and DNA esul s we e included in he main clonali y ana- lysis. Mu a ions called om c DNA sequencing (plasma) we e compa ed wi h mu a ions om issue biopsy DNA sequencing ( umo ). Tumo clonali y was analyzed wi h R package “SciClone”[36]. Non-synonymous “sho ” mu a ions (i.e., missense, non-sense, ameshi , non- ameshi (in/del), p omo e , and splice) we e included in DNA. Used VAF o hese sho mu a ions o ge he mul iple clones o each 37 pai ed pa ien , 26 pa ien s had clonali y analyze. Then we mapped he mu a ions o c DNA o DNA, labeled he same mu a ion genes as ed. Analyses we e pe o med using R e sion 3.3.0. Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 Page 3 o 13 Resul s Pa ien cha ac e is ics The sample was comp ised o 177 pa ien s wi h di e se cance s who had a liquid biopsy nex -gene a ion sequen- cing c DNA es pe o med on hei plasma. Pa ien s’ median age was 65 yea s old ( ange 26–90). Mos we e whi e (83.6%) wi h a his o y o smoking (cu en / ecen : 29.4%; o me >5 yea s: 45.8%). The e was a p edominance o ad anced-s age (84.7%) e sus ea ly-s age (14.2%) can- ce s, and he mos commonly ep esen ed cance s we e lung adenoca cinoma (29.4%), non-small cell lung cance (13.5%), and lung squamous ca cinoma (11.9%) ollowed by head/neck cance (9.0%), colo ec al and cance o unknown p ima y (CUP) (6.8%), panc ea ic cance (3.9%), o he GI (3.4%), small cell lung cance (3.4%), and o he cance s. Table1p o ides hebaselinepa ien s cha ac e is ics. Mu a ions in c DNA and associa ion wi h su i al We de ec ed 628 non-synonymous al e a ions in 61 o he 73 cance - ela ed genes om he Gua dan 360 c DNA es s wi h a mean o 3.55 mu a ions pe pa ien . Twel e genes we e no ound o be mu a ed in he whole sample. Eigh een pa ien plasma samples did no e eal mu a ions in he es (Addi ional ile 1: Table S1). Figu e 1a shows he op 30 mos equen ly mu a ed genes including TP53, KRAS,EGFR,PIK3CA,ERBB2,MYC,andBRCA1.The dis ibu ion o he op 30 mu a ed genes in he majo can- ce ypes is shown in Addi ional ile 2: Figu e S1. We nex examined whe he he o e all mu a ion a e in he plasma o any speci ic gene mu a ions we e asso- cia ed wi h clinical cha ac e is ics. The o e all mu a ion a es in he plasma and he mos commonly mu a ed genes (e.g., TP53,EGFR,KRAS) exhibi ed simila le els in ea ly (I and II) and la e (III and IV) s ages (Fig. 1b). We pe o med a sepa a e analysis o lung cance s (in- cluding LUAD, LUSC, NSCLC-NOS, and SCLC), which ep esen ed he majo i y o ou coho (103 o 172 cases) and ound simila s age-independen mu a ion a es in he plasma (Addi ional ile 2: Figu e S2). We did no ind any speci ic gene mu a ions associa ed wi h ea ly s age cance s. In his pa icula coho wi h a limi ed numbe o ea ly s age cance s, we did no de ec mu a ions in he ollowing genes: ARID1A,KIT,PDGFRA,BRCA2, Table 1 Demog aphics o pa ien s Cha ac e is ic No. (%) Gende Male 96 (54.2) Female 81 (45.8) Age < 55 yea s 38 (21.5) 55–65 yea s 55 (31.1) 65.1–75 yea s 57 (32.2) 75.1–90 27 (15.2) BMI Unde weigh (<18.5) 12 (6.8) No mal (18.5 < =BMI < 25) 74 (41.8) O e weigh (25 < =BMI < 30) 57 (32.2) Obese (> = 30) 34 (19.2) Smoking his o y Cu en / ecen a 52 (29.4) Fo me 81 (45.8) Ne e 44 (24.8) Race Whi e o Caucasian 148 (83.6) Black o A ican Ame ican 24 (13.6) Asian 2 (1.1) O he 3 (1.7) S age S age I 13 (7.4) S age II 12 (6.8) S age III 28 (15.8) S age IV 122 (68.9) Unknown 2 (1.1) # o me as asis si es 0 85 (48.0) 1 64 (36.2) 2 18 (10.2) ≥3 10 (5.6) Vi al s a us Ali e 118 (66.7) Dead 59 (33.3) Tumo ype Lung adenoca cinoma 52 (29.4) Non-small cell lung cance -no o he wise speci ied 24 (13.5) Lung squamous ca cinoma 21 (11.9) Head/Neck 16 (9.0) Colo ec al 12 (6.8) Cance o unknown p ima y (CUP) 12 (6.8) O he 9 (5.1) Table 1 Demog aphics o pa ien s (Con inued) Panc eas 7 (3.9) O he GI 6 (3.4) Small cell lung cance 6 (3.4) B eas 3 (1.7) Kidney 3 (1.7) Li e 3 (1.7) P os a e 3 (1.7) a Recen includes smoke s who qui wi hin he pas 5 yea s Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 Page 4 o 13 FGFR2,ALK,CCND1,PTEN,GNAS,FGFR3,CCND2, and RAF1. The op- anked mu a ed genes ha e been shown o be clinically aluable in designing a ge ed he apeu ics. One g oup o genes ha has gained a en ion is DNA amage epai and ch oma in emodeling genes. The e was a signi ican di e ence in mu a ion load be ween pa ien s wi h BRCA1,ARID1A,BRCA2, and ATM mu a- ions and pa ien s wi hou hese mu a ions (p= 1.047e −09 , Mann-Whi ney-Wilcoxon ank sum) (Fig. 2a). We also explo ed a pa ien diagnosed wi h p os a e cance who exhibi ed a BRCA1 mu a ion in his c DNA. The pa- ien declined u he cy o oxic chemo he apy; he e o e, 28 mon hs a e his ini ial diagnosis o me as a ic p os- a e cance , he s a ed ecei ing ea men wi h he PARP inhibi o olapa ib. 3 mon hs a e ini ia ion o ola- pa ib, imaging e ealed dec ease in he size o mul iple li e lesions, wi h he index lesion measu ing 1.4 cm, om 2.9 cm p io (Fig. 2b). The pa ien esponded well o olapa ib o a o al o 6 mon hs be o e de eloping clinical e idence o disease p og ession. A ecen s udy epo ed ha a mu a ion a e o h ee in he plasma using he Gua dan 360 pla o m was asso- cia ed wi h su i al in a pan-cance s udy simila o ou s [37]. We analyzed he ela ionship be ween mu a ion a es and su i al in ou coho . We ound ha a mu a- ion numbe o h ee did indeed signi ican ly sepa a e he pa ien s in o long and sho su i al g oups. Fu he , we showed ha his ela ionship be ween highe mu a- ion a e and sho e su i al was consis en using all cu o alues om one o six (Fig. 3). We also ound a sligh associa ion be ween mu a ion a es and age and BMI s a us in uni a iable (Mann- Whi ney-Wilcoxon ank sum), bu no in he mul i a i- able es (K uskal-Wallis) (Addi ional ile 1: Table S2). The e alua ion o mu a ion a e and he numbe o si es o me as asis showed ha pa ien s wi h mo e han h ee si es o me as asis had a signi ican ly highe mu a ion a e han pa ien s wi hou me as asis (p= 0.02, Mann- Whi ney-Wilcoxon ank sum). None o he 18 c DNA mu a ion- ee plasma samples, many om la e s age cance pa ien s, had me as asized o mo e han wo di - e en dis an o gan si es (Addi ional ile 1: Table S1). c DNA mu a ions and clinical signi icance o smoking in lung cance WFBMC is loca ed in Wins on-Salem, NC a he epicen- e o he US obacco indus y. As a consequence, smok- ing ela ed cance s, especially lung cance s, a e a c i ical public heal h issue in ou ca chmen a ea, which consis s o 58 coun ies su ounding Wins on-Salem and ex end- ing in o neighbo ing s a es. In ou liquid biopsy p ojec o P ecision Oncology Ini ia i e, 103 o he 177 pa ien s we e diagnosed wi h lung cance , including adenoca cin- oma (LUAD), squamous ca cinoma (LUSC), non-small cell-no o he wise speci ied (NSCLC-NOS), and small cell lung cance (SCLC) (Table 1). An analysis o gene mu a ions among he lung cance subg oup showed op anked mu a ions as TP53,KRAS,EGFR, and PIK3CA (Fig. 4a). The e is a gene al consis ency among he op en mu a ed genes be ween Wake Fo es pa ien s and h ee [22, 37, 38] o he published lung cance coho s ab Fig. 1 Global landscape o c DNA mu a ions. aGlobal c DNA mu a ional landscape o all pa ien s o he op 30 genes ha ing he la ges ac ion o mu a ions. Top and le ba cha s show he numbe o mu a ions and pe cen o mu a ed samples, espec i ely. Lowe pa o panel A summa izes clinical in o ma ion om each pa ien . bc DNA mu a ional landscape o pa ien s’s age known o he op 30 genes ha ing he la ges ac ion o mu a ions. Two pa ien s’s ages a e unknown Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 Page 5 o 13 (Addi ional ile 1: Table S3). The no able di e ence is ha Myc and BRCA1 mu a ions a e among he op en mu a ed genes only in pa ien s seen a Wake Fo es . The e a e 34 cu en / ecen smoke s and 53 o me (>5 yea s) smoke s, and only 16 ne e smoke s among he lung cance sample. We de ec a highe mu a ion a e in ne e smoke s han in cu en / ecen and o me smoke s (Fig. 4b). Fu he , compa ed o cu en / ecen (23.5%, 8/34) and o me smoke s (26.4%, 14/53), mu a- ions in EGFR and ERBB2 we e signi ican ly highe in ne e smoke s (68.8%, 11/16; EGFR:p= 0.12, ERBB2:p=0.02, EGFR,andERBB2:p= 0.005, Fishe ’s exac es ). Mu a ion numbe in he plasma is also co ela ed wi h su i al in lung cance cases: a highe numbe o mu a- ions is associa ed wi h poo e su i al (Addi ional ile 2: Figu e S3). Like he o e all coho , mu a ions in TP53, EGFR, and KRAS we e de ec ed in bo h ea ly and la e s ages lung cance s (Addi ional ile 2: Figu e S2). Gi en hese indings, one may hypo hesize ha he agg essi e CTCs en e ci cula ion a an ea ly s age in lung cance de elopmen when hese cells likely ep esen a mino popula ion o he whole umo . Compa a i e analysis o he c DNA and DNA mu a ion esul s To es whe he CTCs as ep esen ed by he mu a ed genes en e ci cula ion du ing ea ly s age lung cance , we pe o med de ailed compa a i e analyses o mu a ion esul s in 37 lung cance cases ha had bo h umo sequencing esul s by Founda ionOne es and plasma sequencing esul by Gua dan 360. The esul o conco d- ance es shows he pe cen age o op mu a ed genes in c DNA and DNA o hese 37 lung cance pa ien s (Addi ional ile 2: Figu e S4). The wo es s we e pe - o med on samples a di e en collec ion ime excep o wo cases. T ea men s we e gi en o some cases be ween he wo collec ion ime poin s. We hus desc ibe he cases based on he umo s age and he sequence be- ween Founda ionOne es (F) and Gua dan 360 es (G) and whe he he e was ea men (Tx1) o no ea men in be ween (Tx0) (e.g., F-Tx1-G). The umo he e ogenei y is e lec ed by he a ian allele ac ion (VAF) based on F1 es in he X axis in Fig. 5. Among hese 37 cases, 26 we e success ully analyzed o p esen clonali y endpoin . In Fig. 5, each ci cle ep esen s a gene mu a ion. The mu a ions also de ec ed in plasma by Gua dan 360 es a e indica ed by ed ci cles. Fo he s age I cases (n= 2), TP53 and ERBB2 mu a ions we e ound in a mino allele in he umo s wi h VAF less han 20%. Bo h mu a ions we e de ec ed in he plasma. Fo he s age III pa ien s (n= 4), TP53 and KRAS mu a ions we e ound in ei he mino (<25%) and majo (>25%) popula ions in umo issues, and bo h could be de ec ed in he plasma. Fo he s age IV pa ien s (n= 20). Two cases had bo h umo and blood collec ed in he same day. Bo h cases we e posi i e o ALK usion in bo h es s, and ALK usions we e p esen in he majo popula ion in he umo . Fou cases had blood d awn be o e su ge y. Mu a ions o a b Fig. 2 DNA damage epai (DDR) and ch oma in emodeling gene mu a ions a e associa ed wi h inc eased mu a ion numbe and may sensi ize umo o PARP inhibi o . aPa ien s wi h highe DDR and ch oma in emodeling gene mu a ions numbe s (n= 42) compa ed wi h pa ien s wi h lowe mu a ion numbe s (n= 135). The black do ed line ma ks he median o he dis ibu ion. *** (p< 0.001), Mann-Whi ney-Wilcoxon ank sum. bComposi e image o axial con as -enhanced compu ed omog aphy (CT) slicing h ough he li e demons a es a p og essi e dec ease in size o wo hepa ic me as ases p io o ea men (le ), 3 mon hs (middle), and 5 mon hs ( igh ) a e he ini ia ion o olapa ib he apy Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 Page 6 o 13 known d i ing mu a ions we e ound in mino popula- ions in some cases, and hese mu a ions (e.g., TP53 in pa ien 157, PIK3CA in pa ien 86, NF1 in pa ien 132, and CTNNB1 in pa ien s 104 and 159) we e de ec ed in plasma (Fig. 5). Simila pa e ns we e seen in he cases whe e blood was d awn a e su ge y and in a numbe o he cases a e he adju an chemo he apy o adia ion he apy (Tx1). In wo such cases, mu a ions o PDGFRA (in pa ien 79) and BRAF (in pa ien 131) ound in he mino popula ions in he umo s we e s ill p esen in he blood e en a e adju an he apy pos -su ge y sug- ges ing hey ep esen esis an clones (Fig. 5). These wo pa ien s could be candida es o a ge ed he apy using speci ic d ugs o he mu a ed p o eins. Moni o ing o lung cance p og ession by Gua dan 360 es Mu a ions in EGFR ep esen one o he mos common ac ionable a ge s o cance ea men [39, 40]. A num- be o gene a ions o inhibi o s (e.g., e lo inib, ge i inib, Fig. 3 Highe mu a ion numbe s in he c DNA is associa ed wi h dec eased su i al. Highe mu a ion numbe s in c DNA is associa ed wi h poo su i al. nde ines he numbe o mu a ions, and su i al plo s a e sepa a ed by mu a ion numbe s: n= 1, 2, 3, 4, 5, and 6 mu a ions. Blue lines indica e mo e han nmu a ions, and he pink lines indica e equal o o less han nmu a ions. P alues we e de i ed using he log- ank es Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 Page 7 o 13 a a inib, and osime inib) ha e been de eloped o a ge speci ic EGFR mu a ions ha a e equen ly ound in un ea ed lung cance and ecu en lung cance s [41–47]. The e o e, lung cance managemen is known o bene i om longi udinal sampling, moni o ing, and selec ion o di e en inhibi o s [48]. The ac ha EGFR mu a ions can a y o e ime u he highligh s he signi icance o longi udinal moni o ing o mul iple plasma samples du - ing he disease p og ession and pos - ea men . In ou lung cance pa ien s, i e pa ien s we e ollowed wi h his app oach (pa ien no. 1–5inFig.6). Ini ially, he EGFR del 19 was de ec ed in he o iginal issue biopsy o pa ien 1 based on PCR es ing pe - o med by ou molecula pa hology labo a o y bu had no EGFR del 19 mu a ion by Gua dan 360 es ing (le panel o pa ien 1, Fig. 6). The pa ien ecei ed ini ial ea men wi h e lo inib and expe ienced a pa ial e- sponse wi h con ol o he disease o 7 mon hs. The pa- ien had disease p og ession, and he Gua dan es ing o he c DNA a p og ession indica ed he eme gence EGFR T790M, and EGFR del 19. The pa ien was hen ea ed wi h he T790M inhibi o , osime inib, and expe ienced apid clinical and adiog aphic imp o emen . He ini ial ollow-up CT scans indica ed he p esence o mo e scle o ic-appea ing bone me as ases. He apid clinical im- p o emen a e beginning osime inib ea men co e- sponded o umo la e in he ecen CT scan image. Tumo la e is a clinical en i y which is only obse ed in highly ea men -sensi i e cance s. Subsequen imaging con i med ha hese lesions ep esen ed la e, and she con inued o espond o osime inib o 6 mon hs. Pa ien 2 was success ully being ea ed wi h e lo inib and was ound o ha e EGFR del 19 mu a ion and TP53 mu a ion in he ini ial plasma sample. This pa ien e en- ually p og essed on e lo inib and was ea ed by adi- a ion and chemo he apy as well as immuno he apy. The second plasma sample was sen o in es iga e eme gence o T790 du ing a pe iod o p og essi e disease bu had no de ec able mu a ions by Gua dan 360 es ing. Pa ien 3 had EGFR L858R, S811F, and copy numbe ampli ica ion al e a ions in he i s es . A e ea men wi h e lo inib o 2 mon hs, he e was a signi ican a b Fig. 4 Gene mu a ions in lung ca cinoma a e associa ed wi h smoking s a us. aMu a ional landscapes o lung cance s showing 30 o he mos equen ly mu a ed genes. Top and le ba cha s show he numbe o mu a ions and pe cen o mu a ed samples, espec i ely. bEGFR and ERBB2 gene mu a ions concen a e mainly in ne e smoke s Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 Page 8 o 13 Fig. 5 TP53,PDGFBA,BRAF,ERBB2,CTNNB1,EGFR, and ARID1A mu a ions p esen in mino clones in he p ima y umo s a e de ec able in plasma c DNA. The X axis ep esen s allele a ian ac ions. Each ci cle ep esen s one gene mu a ion p esen in umo issues as examined by Founda ion1 es (F). The cases p esen ed mani es he e ogenei y and mul iple clonal cha ac e is ics. Mu a ions also ound in Gua dan 360 es (G) a e indica ed by ed ci cle. The o de o he wo es s and whe he he pa ien was ea ed (Tx1 o yes and Tx0 o no) is shown in he igh panel Yang e al. Jou nal o Hema ology & Oncology (2017) 10:100 Page 9 o 13