Integrated clinical, whole genome, and transcriptome analysis of multisampled lethal metastatic prostate cancer

In eg a ed clinical, whole-genome, and ansc ip ome analysis o mul isampled le hal me as a ic p os a e cance G. S e en Bo a, 1,3 Heini M.L. Kallio, 1,3 Ma i Annala, 1,3 Ka i Ki inummi, 1 Gunilla Högnäs, 1 Se gei Häy ynen, 1 Tommi Ran ape o, 1 Vi pi Ki inen, 1 William B. Isaacs, 2 Teemu Tolonen, 1 Ma i Nyk e , 1 and Tapio Visako pi 1 1 P os a e Cance Resea ch Cen e , Ins i u e o Biosciences and Medical Technology, BioMediTech, Uni e si y o Tampe e and Fimlab Labo a o ies, Tampe e Uni e si y Hospi al, FI-33014 Tampe e, Finland; 2 The James Buchanan B ady U ological Ins i u e, Johns Hopkins Uni e si y School o Medicine, Bal imo e, Ma yland 21287, USA Abs ac We epo he i s combined analysis o whole-genome sequence, de ailed clinical his o y, and ansc ip ome sequence o mul iple p os a e cance me as ases in a single pa ien (A21). Whole-genome and ansc ip ome sequence was ob ained om nine ana omically sepa a e me as ases, and a ge ed DNA sequencing was pe o med in cance ous and noncance ous oci wi hin he p ima y umo specimen emo ed 5 y be o e dea h. T ansc ip ome analysis e ealed inc eased exp ession o and ogen ecep o (AR)- egula ed genes in li e me as ases ha ha bo ed an AR p.L702H mu a ion, sugges ing a dominan e ec by he mu a ion despi e being p esen in only one o an es ima ed 16 copies pe cell. The me as ases ha bo ed se e al al e a ions o he PI3K/ AKT pa hway, including a clonal uncal mu a ion in PIK3CG and p esen in all me as a ic si es s udied. The lis o uncal genomic al e a ions sha ed by all me as ases included homozygous dele ion o TP53, hemizygous dele ion o RB1 and CHD1, and ampli ica ion o FGFR1. I he pa ien we e ea ed oday, gi en his knowledge, he use o second- gene a ion and ogen-di ec ed he apies, cessa ion o glucoco icoid adminis a ion, and he apeu ic inhibi ion o he PI3K/AKT pa hway o FGFR1 ecep o could p o ide pe sonalized bene i . Th ee p e iously un epo ed uncal clonal missense mu a ions (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) we e exp essed a he RNA le el and assessed as d uggable. The uncal s a us o mu a ions may be c i ical o e ec i e ac ionabili y and me i u he s udy. Ou indings sugges ha a la ge se o deeply analyzed cases could se e as a powe ul guide o mo e e ec i e p os a e cance basic science and pe sonalized cance medicine clinical ials. [Supplemen al ma e ial is a ailable o his a icle.] INTRODUCTION Al hough whole-genome sequencing (WGS) has been epo ed om oughly 2500 indi id- ual human cance p ima y umo s o da e (Ding e al. 2010, 2014; Bass e al. 2011; Be ge e al. 2011, 2012; Chapman e al. 2011; Roychowdhu y e al. 2011; Ellis e al. 2012; Egan 3 These au ho s con ibu ed equally o his wo k. Co esponding au ho : [email p o ec ed] © 2016 Bo a e al. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License, which pe mi s euse and edis ibu ion, excep o comme cial pu poses, p o ided ha he o iginal au ho and sou ce a e c edi ed. On ology e ms: malignan geni ou ina y ac umo ; neoplasm o he geni ou ina y ac Published by Cold Sp ing Ha bo Labo a o y P ess doi: 10.1101/mcs.a000752 | RESEARCH REPORT COLD SPRING HARBOR Molecula Case S udies Ci e his a icle as Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 1 o 15 e al. 2012; Go indan e al. 2012; Imielinski e al. 2012; Nik-Zainal e al. 2012; Tu ajlic e al. 2012; Wal e e al. 2012; Welch e al. 2012; Baca e al. 2013; Beà e al. 2013; B as ianos e al. 2013; B ennan e al. 2013; Dulak e al. 2013; Ho e al. 2013; Kan e al. 2013; Mo in e al. 2013; Newbu ge e al. 2013; Pugh e al. 2013; Zhang e al. 2013; B uin e al. 2014; Chen e al. 2014, 2015; Mo ison e al. 2014; Nadauld e al. 2014; Ouyang e al. 2014; Wang e al. 2014; Ande sson e al. 2015; Engle e al. 2015; Coope e al. 2015; Ko ac e al. 2015; Kim e al. 2015; Ya es e al. 2015), only a small numbe o s udies ha e compa ed WGS da a om mul iple me as a ic umo s and p ima y umo s om he same indi idual (Gundem e al. 2015; Hong e al. 2015; Pa ch e al. 2015). The ecen WGS s udy o mul iple p os a e cance me as ases and p ima y umo samples om 10 indi iduals (Gundem e al. 2015) al- lows compa ison o he esea ch alue o WGS s udies con ined o dozens o hund eds o samples o p ima y umo s (“wide”s udies) e sus s udies ha include bo h p ima y and mul- iple me as ases (“deep”s udies). “Wide”s udies ha e begun o es ablish he landscape o mu a ions ypical o a gi en cance and coho o pa ien s, whe eas “deep”s udies allow phylogene ic acing and analysis o ea u es o umo clonali y and e olu ion in esponse o he apy and linked o he me as a ic pheno ype, ea u es ha a guably ha e g ea e implica ions o he de elopmen o e ec i e p ecision ea men o cance han he ca alog- ing o common mu a ions. Combined analysis o p ima y and me as a ic samples om indi idual p os a e cance pa ien s is pa icula ly impo an in ligh o ecen epo s showing ha le hal me as a ic can- ce s can a ise om low Gleason g ade p ima y cance oci in he con ex o highe Gleason g ade oci in he same pa ien (Ha ne e al. 2013) and ha e en ex ensi e needle biopsies may miss he ocus o umo ha gi es ise o le hal disease (Ha ne e al. 2015; Lindbe g e al. 2015). He e we epo he i s combined analysis o ansc ip ome sequence, de ailed clinical his o y, and whole-genome sequencing o p ima y umo si es and mul iple me as ases om a single pa ien (A21), deepening he analysis o his case beyond he WGS da a al eady epo ed in Gundem e al. (2015), and epo ou indings om he poin o iew o pe son- alized cance medicine. RESULTS Clinical P esen a ion and Family His o y Ini ial clinical conce n was aised when he pa ien (A21) was ound o ha e ele a ed se um p os a e-speci ic an igen (PSA) (25.2 ng/mL) a he age o 65 (Fig. 1A). P os a e digi al ec al examina ion a e he PSA es esul s we e a ailable showed a “la ge leshy p os a e, app oxima ely 45–60 g ams, wi h sub le indu a ion loca ed deep wi hin he igh base o he gland. The ma gins we e no mal.”T ans ec al ul asound showed “cys s and calci ica- ions in he cen al zone compa ible wi h benign p os a ic hype plasia, as well as hypoechoic nodules in he pe iphe al zone in he midline, on he igh and he le , which we e suspicious o malignancy. The p os a e capsule appea ed in ac and he seminal esicles appea ed no mal.”P os a e biopsy con ained adenoca cinoma Gleason sco e 4 + 3 = 7. Family his o y was nega i e o p os a e cance . A21’s mo he died o b eas cance in he 80s. Bone scan pe o med 50 d a e biopsy diagnosis was suspicious o bone me as asis; ollow-up adio- g aphs showed degene a i e disk disease, mild os eopo osis, a possible healed ib ac u e, and “no de ini i e e idence o me as a ic oci.”Radical p os a ec omy 3 mo a e p os a e biopsy con ained Gleason 4 + 3 = 7 adenoca cinoma wi h a mino componen o Gleason g ade 5 ca cinoma. The igh pos e io aspec o he p os a e con ained es ablished capsula pene a ion and su gical ma gin posi i e o adenoca cinoma. Seminal esicles, i e igh pel ic lymph nodes, and se en le pel ic lymph nodes we e nega i e o umo . P ecision diagnos ics o p os a e cance COLD SPRING HARBOR Molecula Case S udies Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 2o 15 Figu e 1. Case A21 clinical imeline and pa hology-genomic ela ionships. (A) Clinical e en s and se um PSA plo ed wi h ime in yea s on x-axis. (B) Th ee egions o adical p os a ec omy issue we e mic odissec ed (no - mal s oma, a mixed Gleason 3 + Gleason 5 cance egion, and a egion o ex ap os a ic ex ension con aining Gleason g ade 5 cance ) and compa ed by a ge ed sequencing o whole-genome sequence da a om nine me as a ic si es. Mu a ion pa e ns show ha he wo p ima y cance si es and nine me as a ic si es a e pa o a clonal cance lineage, as e lec ed by acc e ion o mu a ions in SPOP,ASNA1, and PIK3CG and o he s. The mixed Gleason 3 and Gleason 5 p ima y cance egion con ains he ea lies common ances o iden i ied, and cance cells in he egion o ex ap os a ic ex ension a e mo e closely ela ed o cells in all me as a ic si es s udied. All me as a ic si es con ain a PIK3CG p. R472 missense mu a ion ha was no iden i ied in he p ima y cance . The pa e n o sp ead o he me as a ic umo implied om he genomic da a is shown. Numbe s o unique high-con idence soma ic mu a ions iden i ied in me as ases a e lis ed in g ay and ho izon- al lines a e p opo ional in leng h o mu a ion numbe , wi h 1264 mu a ions sha ed by all me as ases, an addi ional 557 soma ic mu a ions unique o A, 178 mu a ions sha ed by all o he me as ases, and so o h. G, Gleason g ade; EPE, ex ap os a ic ex ension; LN, lymph node. P ecision diagnos ics o p os a e cance COLD SPRING HARBOR Molecula Case S udies Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 3o 15 Se um PSA was <0.2 ng/mL, 1.6 y a e ini ial biopsy diagnosis. O no e, 3.8 y a e p os a- ec omy, he pa ien de eloped bone pain, his PSA ele a ed o 969 ng/mL, and a bone scan showed di use skele al me as ases. A lu einizing ho mone- eleasing ho mone (LHRH) ana- log, leup olide, combined wi h an an iand ogen, lu amide, was s a ed. Se um PSA d opped o 0.2 ng/mL and hen ose again o 15.8 ng/mL. This was accompanied by eoc- cu ence o bone pain 6 mo a e beginning leup olide plus lu amide, a which poin he pa- ien began ea men wi h a se ies o co icos e oids (Fig. 1A), as well as es amus ine and inblas ine. Vinblas ine ea men was associa ed wi h ansien dec ease in PSA. O no e, 225 d be o e dea h, lu amide and leup olide we e s opped acco ding o a ailable eco ds. The pa ien de eloped se e e bone pain poo ly con olled wi h opioid and o he pain med- ica ions, and he elec ed o unde go in a enous 89 S adionuclide ea men 46 d be o e dea h om me as a ic p os a e cance . The las measu ed se um PSA le el 64 d be o e dea h was 643.1 ng/mL. The age a p os a e biopsy diagnosis o p os a e cance o subjec A21 has been shi ed by a andomly selec ed numbe be ween −3 and +3 y in acco dance wi h s udy subjec ’s consen o pa icipa e. Clinical alues ha e also been al e ed o educe he isk o eiden i ica ion o he subjec wi hou al e ing clinical meaning. Pa ien A21 was ea ed in he 1990s. All o he a ailable PSA, chemo he apy, s e oid he apy, and adio he apy da a o he pa ien a e shown. A21 was no conside ed o adju- an adio he apy a e adical p os a ec omy, o unclea easons. A21 appea s o ha e a oided e u ning o his physician a e he PSA ini ially ose o 0.9 ng/mL, a e adical p os- a ec omy, un il he de eloped se e e pain. I is also no clea om he a ailable eco ds why leup olide and lu amide we e s opped 225 d p io o dea h, when he was ans e ed o a long- e m ca e acili y. We specula e ha since A21’s p os a e cance was p og essing de- spi e and ogen dep i a ion he apy (ADT) using leup olide and lu amide, his clinicians el i was no longe needed. Whole-Genome Sequencing We pe o med whole-genome sequencing (WGS) in nine and whole- ansc ip ome sequenc- ing (RNA-seq) in six o he same me as a ic cance samples ob ained in a apid au opsy s udy. Phylogene ic Analysis Analysis o soma ic mu a ions ac oss me as ases e ealed he o de o clonal e olu ion om igh ib nodula me as asis h ough se e al in e media e loca ions o ou li e si es in he inal s ages o he disease (Fig. 1B). This was con i med by analysis o DNA ea angemen s and soma ic copy-numbe al e a ions (Supplemen al Figs. 1 and 2). The chain-like ea ange- men s sugges i e o ch omoplexy p esen only in hese li e me as ases demons a e ha majo genome c isis e en s con inue o occu du ing cance p og ession (Baca e al. 2013). All me as ases showed ampli ica ion o he and ogen ecep o (AR) locus (Fig. 2A). AR lo- cus ins abili y was cha ac e ized by ou dis inc pa e ns sugges ing ha AR ins abili y a ose independen ly in he ou ea lies me as a ic si es, consis en wi h sudden s ong selec ion p essu e o main ain and ogen signaling a e onse o ADT (Palmbe g e al. 2000) (see Me hods). We addi ionally ound e idence o a la e-s age AR p.L702H missense mu a ion in all ou li e me as ases (Table 1). The mu an allele ac ion indica ed a single mu an copy o AR in he se ing o ∼16- old AR ampli ica ion (Fig. 2B). The le ad enal me as asis also yielded a single p.L702H ead, bu sequencing e o could no be uled ou (Fig. 2B). Noneo heme as asesshowede idenceo signi ican exp essiono ARc yp icexon a ian s. T ansc ip ome Sequencing RNA-seq in he six me as a ic si es demons a ed signi ican up- egula ion o and ogen- eg- ula ed genes in samples wi h he AR p.L702H mu a ion (Fig. 2C). The p.L702H mu an is P ecision diagnos ics o p os a e cance COLD SPRING HARBOR Molecula Case S udies Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 4o 15 known o ha e inc eased a ini y o glucoco icoids leading o AR ansac i a ion (Zhao e al. 2000; Ca ei a e al. 2014). I a pa ien wi h simila indings we e ali e oday, de ec ion o mu- an AR migh indica e hal ing co icos e oid ea men , wi h po en ial bene i o he pa ien . The indings indica e ha he AR locus can be a ge ed by wo hi s (AR ampli ica ion and Figu e 2. E olu ion o and ogen ecep o (AR) egion X Ch omosome al e a ions by whole-genome and an- sc ip ome sequencing in case A21. (A)AR egional s uc u al ins abili y signa u e in ea lie me as ases ( igh ib, le iliac c es , le cla icle LN, and le ad enal) in he clonal e olu ion map shown in Figu e 1B con ain ou dis inc pa e ns, he la e o which (le ad enal) is sha ed by all subsequen si es on he map, sugges ing in- dependen e olu iona y selec ion o dis inc AR s uc u al esponse o and ogen dep i a ion he apy (ADT) in a leas hese ou s udied independen si es. Whe he hese asso ed AR-mu a ed subclones exis ed in small numbe s be o e selec ion canno be de e mined om he cu en s udy. (B) All ou li e me as ases and he le ad enal me as asis con ained an AR p.L702H mu a ion in one copy o AR. The mu a ion is also p esen in he RNA-seq da a om he same samples. The L702H mu an modi ies he AR ligand binding domain and is known o al e ligand speci ici y, ende ing he p o ein esponsi e o glucoco icoids adminis e ed du ing A21’s ea men shown in Figu e 1A (Zhao e al. 2000; Ca ei a e al. 2014). (C) Li e me as ases con aining he AR p.L702H mu a ion exhibi ed signi ican ly inc eased exp ession o and ogen- egula ed genes KLK3, ACPP, FKBP5,SLC45A3, and PCA3 as compa ed wi h h ee me as a ic si es whe e he AR p.L702H was no de ec ed (∗P< 0.05, ∗∗P< 0.01 and ∗∗∗P< 0.001; see Me hods). P ecision diagnos ics o p os a e cance COLD SPRING HARBOR Molecula Case S udies Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 5o 15 mu a ion), unde sco ing he impo ance o and ogen signaling in p os a e cance p og es- sion. All o he pa ien ’s sequenced me as ases we e nega i e o ETS (E26 ans o ma ion speci ic) usions. Ta ge ed Sequencing To ace he o igin o he al e a ions ound in he me as ases, we pe o med a ge ed se- quencing o h ee oci in he p os a e: a egion con aining mixed Gleason g ade 3 and 5 can- ce , a egion o ex ap os a ic ex ension (EPE) con aining Gleason g ade 5 cance , and a egion o noncance ous p os a e s oma (Fig. 1B). Based on sequence analysis o hese oci, he me as ases mos closely ma ch mu a ions ound in he egion o EPE. Bo h he e- gion o mixed Gleason 3 and Gleason 5 cance and he egion o Gleason 5 EPE con ain a SPOP p.F102C mu a ion (Ba bie i e al. 2012). DISCUSSION Examining he indings in he me as ases a he AR locus oge he wi h he clinical imeline, we specula e ha me as a ic p os a e cance subpopula ion selec ion occu ed in h ee phases. Phase 1 occu ed a he ime o ini ial ADT, when ou independen AR-ampli ied subclones eme ged in ou sepa a e loca ions (Figs. 1A and 2A). Phase 2 is indica ed by he eme gence o an AR p.L702H mu a ion con e ing glucoco icoid esponsi eness (Figs. 1A, 2B,C). Phase 3 occu ed when leup olide and lu amide ea men we e s opped 225 d be o e dea h, when he mos highly and ogen-plus-co icos e oid esponsi e sub- clone apidly eme ged and popula ed he li e (Figs. 1A, 2B). Sha ed uncal d i e mu a ions in me as ases a e a ac i e po en ial a ge s o pe son- alized he apy. In A21, we ound six po en ially ac ionable genomic al e a ions sha ed by all me as ases and exp essed a he RNA le el, including ou poin mu a ions and wo copy- numbe al e a ions (Supplemen al Table 1). The PI3K/AKT pa hway ha bo ed se e al po en- ially ac i a ing al e a ions, including an AKT3 duplica ion and PIK3CG p.R472C mu a ion in all me as ases, and a PIK3CA duplica ion in all me as ases excep he igh ib me as asis. The le ib me as asis addi ionally ha bo ed a p i a e PIK3CA p.Q546K mu a ion (Table 1). Mu a ions a his esidue dis up he PI3KCA helical domain and a e known o esul in PI3K pa hway ac i a ion. PIK3CA encodes a ca aly ic subuni o he class IA phosphoinosi- ol-3-kinase (PI3K) complex, whe eas PIK3CG encodes a ca aly ic subuni o he class IB Table 1. Selec ed missense a ian s based on whole-genome sequencing Numbe o a ian eads/ o al eads Ch Posi ion Re Al Gene AA_Change No mal kidney Righ ib Le iliac c es Le cla icle LN Le ad enal Le ib Single li e 4 Single li e 8 Single li e 2 Mul i li e 13 Ch 3 178936094 C A PIK3CA p.Q546K 0/33 0/32 0/56 0/33 0/60 18/59 0/48 0/66 0/54 0/37 Ch 1 165664551 A G ALDH9A1 p.W89R 0/29 18/29 17/27 16/24 23/37 26/36 22/32 18/29 19/26 21/29 Ch 7 106509420 C T PIK3CG p.R472C 0/35 7/25 11/36 12/42 8/24 12/38 8/19 8/24 7/47 15/38 Ch 13 95735408 C A ABCC4 p.R891L 0/30 13/24 25/44 25/31 16/26 20/35 20/32 18/27 22/33 15/28 Ch 17 47696643 A C SPOP p.F102C 0/35 16/22 7/25 6/18 8/24 14/34 12/20 12/26 16/32 16/29 Ch 19 12849387 C G ASNA1 p.P75R 0/33 8/16 13/29 15/30 6/20 9/25 9/19 10/19 16/38 14/33 Ch X 66931463 T A AR p.L702H 0/19 0/176 0/153 0/164 1/83 0/127 8/132 8/91 5/94 4/112 Re , A21 ge mline e e ence allele; Al , A21 al e na i e allele; AA_Change, p edic ed p o ein amino acid change. P ecision diagnos ics o p os a e cance COLD SPRING HARBOR Molecula Case S udies Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 6o 15 PI3K complex. Bo h class I PI3K complexes phospho yla e PIP 2 lipids o p oduce PIP 3 . PIP 3 lipids bind o he plasma memb ane and p o ide a docking poin o AKT amily p o eins (in- cluding AKT3) and hei ups eam ac i a o PDK1, esul ing in ac i a ed AKT signaling. Al hough he biological signi icance o he PIK3CG p.R472C mu a ion is unclea , he mul i- ple hi s o his pa hway sugges ha he pa ien could ha e bene i ed om PI3K/AKT pa h- way inhibi ion h ough d ugs such as NVP-BEZ235 o AZD5363. Ano he po en ially ac ionable inding was a ocal FGFR1 ampli ica ion sha ed by all me as ases. Abe an ac i- a ion o ib oblas g ow h ac o ecep o s by ampli ica ion o usion has been associa ed wi h oncogenic p og ession in a ious cance s including p os a e cance (A ms ong e al. 2011), and FGFR1 inhibi o s a e cu en ly being es ed in clinical ials (Dieci e al. 2013). Th ee o A21’s clonal uncal mu a ions (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) (Table 1; Supplemen al Table 1) we e exp essed a he RNA le el and we e assessed as po en ially d uggable based on analysis using he D ug–Gene In e ac ion Da abase (G i i h e al. 2013). Al hough hese uncal mu a ions likely ep esen passenge e en s, hey could in oduce physiologic ulne abili ies o small-molecule-based syn he ic le hali y and/o c ea e no el a ge s o immuno he apy. Exp essed clonal uncal mu a ions ha all in o his “po en ially help ul passenge ”ca ego y me i u he analysis o de e mine hei ue alue in ad ancing cance p ecision medicine. Wha added alue did WGSp o idein hiss udy?Would whole-exomesequencing (WES) ha e p o ided simila in o ma ion? WGS was c i ical o unde s anding he clonal his o y o he me as ases, as WES would ha e educed he numbe o in o ma i e mu a ions om 741 o below 20. This numbe o mu a ions would no ha e been su icien o econs uc ing he clonal his o y wi h high con idence. Use o exome sequencing would ha e also ende ed de ailed analysis o he AR copy-numbe al e a ions and hei b eakpoin s impossible. These a e esea ch indings wi h po en ial clinical impo , suppo ing he u he use o WGS in ap- p op ia e ials. None heless, using he WGS da a o simula e WES in each me as asis s udied in his case shows ha 35–43 p o ein-al e ing mu a ions would ha e been iden i ied, 32 o which we e sha ed by all me as ases (including he ou po en ially d uggable p o ein-al e - ing mu a ions in PIK3CG,ABCC4,ALDH9A1, and ASNA1). A ma ix o p o ein-al e ing mu- a ions sha ed by any wo me as ases shows ha he 32 uncal mu a ions could be pe ec ly o nea ly pe ec ly sepa a ed om he non uncal mu a ions by combining esul s o any wo bone me as asis samples o combina ion o one bone and one isce al me as asis samples. This inding should be es ed in u u e s udies (Table 1; Supplemen al Table 3). How does his case compa e o he cases in he ecen clinical exome sequencing in ad- anced p os a e cance s udy wi h 150 pa ien s (Robinson e al. 2015)? Based on Figu e 2 in Robinson e al., Case A21 epo ed he e has in e es ing simila i ies o Case 32 in Robinson e al. These wo cases sha e AR ampli ica ion and loss o bo h copies o TP53; nei he has loss o PTEN o con ains ETS usions; A21 has missense mu a ions in one copy o PI3 kinase co e p o ein PIK3CG; Case 32 has ampli ica ion o bo h copies o PI3 kinase co e p o ein PIK3CB; bo h cases show loss o CHD1 (bo h copies in Case 32, hemizygous in Case A21); and bo h ha e missense mu a ion o one copy o SPOP. Genomic di e ences include al e a ions in BRCA2 in Case 32 only, and loss o RB1 and TET2 in A21 only. FGFR1 is ampli ied in A21, and none is epo ed o Case 32. Whe he hese simila i ies and di e ences a e meaning ul on a biologic o clinical le el would equi e mo e de ailed compa ison using well-cu a ed da- abases and a la ge numbe o cases, bu hey sugges ha he ield would likely bene i om he abili y o do such compa isons. In conclusion, analysis o he “deep”da a se a ail- able o Case A21 p o ides biological insigh s ele an o p os a e cance biology in gene al and o Case A21 in pa icula . These insigh s a e a mo e use ul han wha can ypically be ob ained om “wide”s udies. In conclusion, his case s udy sugges s ha once ini ial selec ion o cas a ion- esis an subclones occu s a e ini ial ADT, and ogen esponsi eness is none heless main ained by P ecision diagnos ics o p os a e cance COLD SPRING HARBOR Molecula Case S udies Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 7o 15 me as a ic cells. Rein oduc ion o and ogen by s opping leup olide and lu amide and con- inuing co icos e oids was associa ed wi h popula ion o he li e by a clone con aining com- bined ampli ied wild- ype and co icos e oid-sensi i e mu an AR. In addi ion, p.L702H AR mu a ion in app oxima ely one o 16 AR copies pe cell was associa ed wi h ac i a ion o an- d ogen- esponsi e elemen s as e idenced by RNA-seq. Impo an ly, his s udy adds o p e- ious epo s ques ioning he alidi y o molecula ep esen a ion p o ided by andom biopsies o he p ima y umo . To ind he bes po en ial no el d ug a ge s o indi idual pa- ien s, s udies a e needed o de e mine op imal sampling egimes, including “liquid”and me as a ic issue biopsies a di e en ime poin s, o iden i y uncal changes, and o de ec he apy-induced changes. S udies ully in eg a ing he en i e clinical imeline, pa hology, and genomic indings in indi idual pa ien s a e essen ial o meaning ul p og ess in unde - s anding cance and can each he combined esea ch and medical eam o iden i y po en- ially ac ionable al e a ions. We a gue ha such “deep”s udies should become he ule, a he han he excep ion. Finally, iden i ica ion o no el “p ecision”d uggable a ge s is jus a i s s ep. Cu en ly many pa ien s a e no eligible o ecei e iden i ied a ge ed d ugs because o limi a ions in ial and eimbu semen mechanisms. New clinical ial and d ug ap- p o al and eimbu semen mechanisms a e needed o allow he cance p ecision medicine concep o ind i s app op ia e use in medical p ac ice. METHODS Tissue Mic odissec ion and DNA Isola ion DNA and RNA isola ion om he eshly ozen c yos a -mic odissec ed me as ases (n=9; Fig. 3B) was pe o med wi h phenol–chlo o o m ex ac ion o QIAGEN column ex ac ion. A noncance ous kidney sample was used as ge mline e e ence con ol. Cance cell ac ion (CCF) es ima ed by isual analysis o hema oxylin and eosin–s ained dissec ed umo sec- ions and om examina ion o allelic ac ions was be ween 75% and 90% (mean CCF was 84.6%) o all umo samples. WGS was pe o med o all nine me as a ic samples and RNA-seq was pe o med in six o he same me as a ic issue ma e ials (al e na ing sec ions used o DNA o RNA isola ion). Lase mic odissec ion o selec ed oci (Fig. 1B) om he p os a e emo ed 5 y be o e dea h was pe o med using A c u us and MMI dissec ion sys- ems. DNA isola ion was pe o med using QIAGEN FFPE ki s. Whole-Genome Lib a y Cons uc ion and Sequencing Genomic DNA was shea ed in o 500-bp agmen s using a Co a is E210 ul asonica o . O e hangs we e con e ed in o blun ends using T4 DNA polyme ase and Klenow enzyme. An adenine was added o he 3′end o he blun phospho yla ed DNA agmen s, and adap - e s we e liga ed on bo h ends. Liga ed p oduc s we e pu i ied by aga ose gel elec opho e- sis ollowed by QIAquick gel ex ac ion, o emo e esidual ee and sel -liga ed adap e s and o selec p ope ly sized empla es o clus e gene a ion. DNA agmen s wi h adap e s on bo h ends we e ampli ied using wo p ime s ha annealed o he adap e s. Polyme ase chain eac ion (PCR) p oduc s we e checked and pu i ied by aga ose gel elec opho esis. The agmen size and mola concen a ion o each lib a y we e de e mined using he Agilen 2100 Bioanalyze and ABI Real-Time PCR Sys em (S epOnePlus), espec i ely. As mean agmen size inc eased o 622 bp a e adap e liga ion, agmen s be ween 600 bp and 684 bp we e selec ed. An Illumina Clus e S a ion was used o hyb idize samples on o a low cell and ampli y hem o sequencing on Illumina HiSeq 2000. Raw image iles we e p ocessed by Illumina pipeline o base-calling wi h de aul pa ame e s esul ing in 90-bp pai ed-end eads. Reads wi h oo many N bases (>10%) o low base quali y (>50% P ecision diagnos ics o p os a e cance COLD SPRING HARBOR Molecula Case S udies Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 8o 15 bases wi h base quali y <5) we e disca ded. Lib a y cons uc ion and sequencing was pe - o med a BGI (Hong Kong). A e age whole-genome sequence co e age was 30× o can- ce ous and noncance ous samples. Median co e age o coding egions was 23× o highe in all samples (Table 2). The ange o co e age a di e en dep hs o each umo sample is shown in Supplemen al Figu e 4. Ta ge ed Sequencing To alida e mu a ions ound by whole-genome sequencing and o sequence mic odissec ed egions om he p ima y umo , a ge ed sequencing o 88 amplicons (a e age 150 bp in leng h) was ca ied ou using Illumina MiSeq and an Illumina T uSeq Cus om Amplicon ki designed using Illumina Design S udio so wa e. O he 88 amplicons, 63 amplicons we e a ge ed a soma ic mu a ions, i e a ge ed a ge mline single-nucleo ide polymo phisms (SNPs) we e used as posi i e con ols, and he emaining 20 we e used o assessing copy numbe . The lis o 63 soma ic mu a ion a ge s included 17 p o ein-al e ing mu a ions obse ed in all me as ases, 16 p o ein-al e ing mu a ions obse ed in a subse o me as ases, and 30 non-CDS (coding sequence) mu a ions ha we e alida ed because hey we e in o ma i e wi h ega d o he p og ession model (Supplemen al Table 2). O he 63 a ge ed mu a ions, 60 yielded eads, and 57 o 60 (95%) yielded geno ype calls iden ical o hose ob ained om whole-genome sequencing. Th ee a ge ed mu a ions yielded low mu an allele ac ion in all samples (including he no mal issue) and we e eca ego ized as sequencing e o s. Amplicons used o copy-numbe assessmen included i e amplicons each o TP53, RB1,CHD1, and he 23-Mb egion in Ch omosome 2. These ou egions we e dele ed in all me as ases, and he amplicons we e included o assess hei copy numbe in he p os a- ec omy sample. Whole-T ansc ip ome Lib a y Cons uc ion and Sequencing RNA isola ed om se en o he me as ases unde wen ansc ip ome sequencing. Beads wi h oligo(dT) we e used o isola e poly(A) mRNA a e collec ion o o al RNA. F agmen a ion bu e was added o shea mRNA in o sho agmen s and o syn hesize he i s -s and cDNA wi h andom hexame p ime s. The second-s and cDNA was syn he- sized using bu e , dNTPs, RNaseH, and DNA polyme ase I, espec i ely. Sho agmen s Table 2. The co e age o he whole-genome and whole- ansc ip ome sequencing Sample Whole-genome sequencing Whole- ansc ip ome sequencing To al eads Aligned (%) Median CDS co e age To al eads Aligned (%) m RNA (%) Righ ib 1,200,343,314 93 26 108,387,928 68 15 Le iliac c es 1,379,274,636 94 37 105,250,920 55 10 Le cla icle LN 1,126,637,546 93 31 110,086,613 91 82 Le ad enal 1,151,593,908 93 27 Le ib 1,252,845,806 93 30 110,678,869 73 8 Single li e 2 1,481,140,684 91 38 108,970,487 93 9 Single li e 4 1,077,269,004 92 23 Single li e 8 1,164,407,226 92 24 11,237,2452 88 12 Mul i li e 13 1,088,762,606 92 28 113,997,841 93 10 No mal kidney 1,169,821,684 91 34 CDS, coding DNA sequence; m RNA, mi ochond ial RNA. P ecision diagnos ics o p os a e cance COLD SPRING HARBOR Molecula Case S udies Bo a e al. 2016 Cold Sp ing Ha b Mol Case S ud 2: a000752 9o 15