AU2015203075A1 - Biomarkers for determining sensitivity of breast cancer cells to HER2-targeted therapy - Google Patents

Description

BIOMARKERS FOR DETERMINING SENSITIVITY OF BREAST CANCER CELLS TO HER2-TARGETED THERAPY The present application is a divisional application of Australian Application 5 No. 2010248884, which is incorporated in its entirety herein by reference. CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 611178,458, filed May 14, 2009, U.S. Provisional Application No. 61/180,787, filed 10 May 22, 2009, U.S. Provisional Application No. 611187,246, filed June 15, 2009, U.S. Provisional Application No. 61/228,522, filed July 24, 2009, U.S. Provisional Application No. 61/235,646, filed August 20, 2009, U.S. Provisional Application No. 61/241,804, filed September 11, 2009, U.S. Provisional Application No. 61/262,856, filed November 19, 2009, and U.S. Provisional Application No. 61/265,227, filed 15 November 30,2009, the disclosures of which are herein incorporated by reference in their entirety for all purposes. BACKGROUND OF THE INVENTION [0002] The process of signal transduction in cells is responsible for a variety of biological functions including cell division and death, metabolism, immune cell 20 activation, neurotransmission, and sensory perception to name but a few. Accordingly, derangements in normal signal transduction in cells can lead to a number of disease states such as diabetes, heart disease, autoimmunity, and cancer. [0003] One well characterized signal transduction pathway is the MAP kinase 25 pathway, which is responsible for transducing the signal from epidermal growth factor (EGF) to the promotion of cell proliferation in cells (see, Figure 1 of PCT Publication No. W02009/108637, the disclosure of which is herein incorporated by reference in its entirety for all purposes). EGF binds to a transmembrane receptor-linked tyrosine kinase, the epidermal growth factor receptor (EGFR), which is activated by the binding 30 ofEGF. The binding ofEGF to EGFR activates the tyrosine kinase activity of the cytoplasmic domain of the receptor. One consequence of this kinase activation is the autophosphorylation ofEGFR on tyrosine residues. The phosphorylated tyrosine residues on the activated EGFR provide a docking site for the binding ofSH2 domain containing 1 adaptor proteins such as GRB2. In its function as an adaptor, GRB2 further binds to a guanine nucleotide exchange factor, SOS, by way of an SH3 domain on GRB2. The formation of the complex ofEGFR-GRB2-SOS leads to SOS activation of a guanine nucleotide exchange factor that promotes the removal of GDP from Ras. Upon removal 5 of GDP, Ras binds GTP and becomes activated. la i{lt0t41 Following activation, Ras binds to and activates he protein kinase activity of1 RAF insa srinethreonine-secific protein kine. What follows is the activation of a protein kinase cascade that leads to cell proliferation, In outline, RAF kiase then phosphorylates and activates MEK, another serinedtheoine kinase. Activated MEK phosphorylates and > activates mnitogen-activated protein inase (MAPK}. Among the targets tor tordher phosphorylation by MAAPK are 40S ribosomal protein S6 kinase (RSK)9. The ph osphorylat ion of RSK by MAPK results in activation of RSK, which in tun phsphoglates ribosomal prti E6, Another known target of MAPK is the proto-oncogene e-Mye, gene important fIr cell proliration which is stated in a variety of cancers, MAPK also phrosphotyiate 10 and activates another protein kinase\ MNK, which in tun phosphorylates the transmcpion factor, CREB. Tndirecty, MAK also regulates the tanscription ofthe Fas gene, which rvncAdes yet another transcription factor tiohed in ce prolierati By altering the levels and activities of soeb tninscription sectors, MAPK transduces the original extracellultar signal from EGF into altered transcription of genes that are important or cell cycle progreson. 15 {M016 Gi ven the central role that signal transduction pathways play in. cell growth, it is not sutprisirg that many cancers arise as a result of mutations and other alterations in signal transduction componets that resnit in aberrant, activation of ce proliferation pathways. For example, overexpression or hyperactiviy of EGR has been associated with a unmer of cancers, including giobastoma multifonne, colon cancer and lung cancer. Th as 20 prompted the development of anticancer therapem;ics directed against EGFR, inciding getitinib and. erlotib for lung cancer, and cetuximab for colon cancer. 0006] Ceximab is an example of a moncinal antibody inhibitor, which hinds to the extace~unar ligand-binding domain of EGER. thus preventing the binding of Iigands which activate the EGFER tyrosine kinase, In comtast, gefitinih and eriotinib are small molecules 2$ which inhbit the intmoelhd located E(FR tyrosine kinase. i the absence of kinase ity, EGR is unable to undergo autophosphoryiatio at tyrosime residues, which isa prerequisite for binding of downstream adaptor proteins, such as GiRB2, by halting the siang cascade in ceis that rely on this pathway tor growth, mmor proliferati and mr o diminished. 30 [O1 71 AddioAdly, other studies have shown hat about 70% oi'han melanoma as and a smaller fraction no ter tamors have a point mutation t599E) i e Raf gene which leads to pscristat actdaion of> te MAPK pathway (Ise eg. Dave eat, Nae 47 9-95 2 (2002)) Such retulia suggest that tnnannai parwtdht signal iansductiton pathways zny be chameteistieoparticula tpes tumoraad hat such specific, altered signa transduction pathways nay be a pmAisigo target forem ea c [**Sj) Given that different cancer treatments, particuily caner chemotherapy, ay 5 fun~ctiosn either directly or indirectly by means of either blocking or activating cellular signal transduction pathways that are involved in cell proliferation or death respectnveiy, the activity~ of a given signal transducton pathway in a particular formn of cancer mnay serve as a good indicator of te efbfiacy ofriu cancer treatmients, Acodinly, in addition to fulfilling other needs, the presnt invention provides a method for evaluatig the I D effctiveness of potential anticancer therapies sor an individual patient, As suelA the present invention provides methods fot assisting a physician in selectong a suitable cancer therapy at the right dose and at dhe right time for every patient, BRWFF SiMMARY OF THE INVENTION [00091 The present invention provides composions and methods for detecting the status IS (eg, expressin and/or activation levels) of components of signal transduction pathways i trumor eel V (a g. cir-culating cells of a breast tumorr. Information on the' expression and/or acdvatnon states of components of signal rTansduction patrhvays derived from practice of the resent invention can be used for cancer diagnosis, prognosis and in the design of cancer treatments. 20 (001 In panicular aspects the present invention provides secular market (bomarkers tt enable the determination or prediction of whether a particular cancer can. respond or is kyo a R co ud (e.g. a HER2 inahihitot As described herein, it has been surprisingly found that borarket in the HER2 pathway such as HER2 and p95HER2 ar'eparticularly usefud in determining or predicting the sensitivity of 25 cells such as breast cancer cells to coounads that modulate $R2 activity (ea. ionoclonal antibodies, tyrosineC kinase uubitor, and the likeV [DU In omeaspect, the present invent provides a method kr determting or predicting the sensitivityaf a c dli to a compound hat modulates KERZactiittheethod comprising (a) contacting the cell with the compound~ 30 (U) ysig the cell.to produce a ellular eta (c;) deternin theexresiont and/ active to (a gkposphorylation) level of one or meore components of a IiER2 signaling pathway in the celdiniiar exrc;and (d) compaing the expressin anwor activation level ofthe one or rore components of the ER2 signaling pathwayv dctermied in step (c) to a refeence xprssion ador activationm level of the oneO or more components of the H WR2 signaling pathway, wherein a difference betweca the exrpression antkr activationt level of the one or more comtponents of the JA.ER2 signaling pathway detennritaed in step (c) and the reference S expression. and/or activation level of the one or more componens of the HER2 signaling pathway indicates that the cel is 'asitve or resistant (te. not sensitive) to the compound. [0012) In preferred aspects, the present invention provides a method for deternidng or predicting the sensitivity of a cell to a comnpond that modulates HIER2 activity, the method comipriing: 15 (a) coating the cell with the cornpoimd; (bt) iysing. the cell to produce ace arxta; (c) dtermdnng the activation level of HIER2 or p95HER2 in the cellular extract; and (d) comparing the activation level of HIE R(2 or p95HER2 detemniined in step 20 (c) to a reference activation level of HER2 or p95HER2, wherein the presence of a higher level of E12 or p95110E activation in the cellular exract compared to the reference activation level of HER2 or p95HER2 cates that the cell is not sensitiv (le.. resistant) to the compounds. fOil 3~In some cenoodiments. the methods ofthe present iventdon amy be useful to aid or 25 asistin de ninag o pre&d gte es flvy of a cell to a compound thatnaidnlates HER2 atvity. In otherembodbneits he methods of th. presentiveenton nay be uasefA Simproving the detenniation or predictioof the senAvityof a cell to a campond hat mrodtlatesNAEP activity 140 In other aspecihe present invention provides a neod for predicting the 30 respond of a tumor to a aOmpound tht modulates HER2aciv the method comprin (a) contacting a cell obtained fnat the tutnor with) the conptmnd; i)lysing the cell to produce a celular extract 4 dcI ueteranBIg thryes~aan anor' aation a pospthadoflj of one more components ofa HR2signalingpathway in the cellular extret and i conepadnt the expresson an/or activato ee ofeone or o e S ~components of the THER2 signaling pathwaye de'rmined in step (c) o a refmcr~e predOn andll/Or actio evel of tIe one or rMOM components ofthe HER2 signaling paithway wherein a ditTerence between hO exprosso and/or activation level of the one or more components oftheHER2 signaing path y deotnOed o step (c) and the rfrene 10 expression and/or activation level of the one or more comnents of the HER2 signaing pathway indicates that the tumor is or is not likely to respond to the compound (e g Ohe moor hs an increased or decreased Ilkelhuood of response to the compound). (001t5J in preferred aspects, the present invention provides a method for predicting th response of a tumor to a compound that modales HER2 activity, the method comparing: (a) contactng a ecli obtained from the tumor with the compound; (b) lysing Ire cell to prosducet a ellular extra;t (c) detenmining the activation level otf{HER2 or p95RER2 in the elihar extract; and (d) comparing the activation ' 'f HER2 or op5ER2 determined in step 20 (c) to a reference activation level of HER2 or p95HER2, wherein the presence of a igher level of ER2 o p9511ER2 activationm the cellular extract compared to the reference activation level of 11ER2 or p95Hi-ER2 indicates that the tumor is not likely to respond to the compound (g, the turnor has a decreased likelihood of response to the comipound}. 25 ( 1]| uono en1bodimnrts the methods of he present invetron mahe useful toaid o assist in predicting the response c' F a tumor to acmpoundtaiodulates HER2 aotit other bdiments, the methods of the present nverin may be anfid to improving the prediction of the response of a trrnr to a compound thatnnochades HER2 aetivity. {Q67 in yet another apet, the present invention proiddes a niettod aonitoring te 30 pome to dwe y th a.compmed tt moidate; HER2 activity in a subject having a tumor and reving therapy with theoampoundthe method comtia: (a) lysing aceul obtained from the tamor to pwoduce cc diar extact; (b) dtrmining the depression and/or ativation (eg., phosphorylation) level o one or moWt components of a HER2 signaling pahway in the cell alar exact and (c) conmparing thre expression and/or activation level of the one or more components o the HER3 sigala g pathway determined in step (b) to a refennece expression and/or activation level of the one orumore components of the HER2 signaling pathway wherein a difference between the expression and/er activation tevel of the one or moe components of the HER2 signaling pathway determined in step (b) and the referee 10 expression and/or activation level of the one or more components of the ER2 signaling pathway indicates hat therapy with the compound should be cntnued or ad ousted (eg. maintain the current dose of the compound, changing a suhsequent dse ofthe compound or rel etig an ahiemative anticancer drag). t018 In preferred aspects the present invention provides a method for mtonitorirng the 1 response to therapy with a coipound that modulates HER2 activity in a subject having a mmor and receiving thempy with the compound, the method comprising; (a) lysing a cel obtained fr the wmor to produce a cellular exact (b) te mining the activation level of HE2L or p95HE2 in the celular extract and (c) comparing the activation level of HER? or pER2 dhenrined in step (b) to areference cilvatio. level of HER2 or p95HER2 wherein the presence of a higher level of HIER2 o; p95HR2 aciao i the cellar extract compared to the reference activation levei of HER2 or p95HER2 indicates that therapy with the con pound should he adjusted (eg, changing a subsequem dose cf the compound or selecting an ahtenative anticancer drug) [(I19} in. some embodiments, the methods of the present invention may be useMi to abd or assist in monitoring the response to therapy with a compound that modslates HER2 acdvity. La other embodinents the methods of the prseAt invention may be useful for providing a prognosis of the response to therapy with. a compound that modulates HE2 activity. 30 [002) In ai frtor aspect. the present invention provides a method for monitoring the HE2 ttus of a subject 'with an initial HlER2-negative primary breast tumor, the method comprising 6 determining dhe WRZstatus of choating tellsoftasodid tttuner obtanied ioma the subject by detecting dte p ore of activated FER2 in the ceulating ces wherein the presence ofa ativated H-1ER2 in the circuating ells indicates a cons on OS a egative satus ot the subject a H2apostive statusu 021l in some emabodimnats the methods of the present inveion may be usefd to aid or assist in monitoring the HR2 status of a sutlect with an initial IER-negive primary breast tumor, In other embhodiments. the methods of the present invemi on may be useful for providing a prognosis of a subjet with an initia! HER2-negatiw primary bret tamor by deteiUMning the HER2 status of the subject in circulating cells of a solid tumor TO [OI22j In an additional aspect the present invention proves a method f selecting a a able anticancer dru for the treatment ofa breas nr the meihod compodig (a)contactng a celbtainedfon aine needle sprate(NA) sample of the tumor with ani antie0ner drug; (b) ysing the cell to produce a ceilula extract; 1 5(c) detennmnn the expression and/or activation level of one or more signa transdnuton molecules in the cellular exrtract; and (d) company the expression and/or activation lvel of the one or more signal transduction maohtlele determined in step (c) to a. reference expressing and/or activation level of the one or o raI transduction molecules, :20 wherein a dit'erence between the expression and/or activate level of the one or more signal transduction molecules determined in step (e) and t he ref erene~ expression and/or activation level of the one or mioret signal transduction molecules indicates that the anticancer dtog is suitable or unsuitable for the treatmientx of the breast tumor, V04231 in soue embodiments, the methods of the present inetion may be useful aid or 2 assist in te selection of a suitable anticancer drug for the treatment of a breast tumor in other cumbodi.ets the methods o preen iv'tionvmy be ueful for improving the select inn of a suitable anticancr drug fo r the treatment of a breast tunor, {00241 Other objects, features, and advantages of the present invention will be apparent to one of 4kill in the art from the following detailed description and figures, RI0DRsCRITON oF T DRAWIN{O 0251 lFgure 1 shows an exenpary samyae processingflovchart fr the isoladon of CTCs from collectedhole bl samples {(I264 Figur 2 shovuthe Verdex CTC eueration resnhs tbr all cancer samples. Left 4 atead agents. Vridex conAting I27 Fire3 shows a aun nmary of EfR and HER2 phospbhttion observed in C positive sames using the proxcltuy assay deserbed here {0029] Figure shows xen gtFNA amodesfw different types of breast cancer us ng 0 Q0030] Figre shows komt dassueTNA model where the activatkin of HJR2 receptor detected using the prmxi assay described herein conrda with uanor IC se. f{1S1 igre (lft shwsa srinm '~~ tthe levls f acwaed- FERR and 1WR2 in PNIA sample fom breast cance tisue with known ar unknowr HERCHE status and nutu a t gw aphica sain of pEGFR and HER2 es in FNA 13211 Figure: 1$ Amow a.ttaina&yssoadER.VveI n.NAsmle.wt ih1 10033 Flgure I shows the detection of pEGFR aid pHER in FNA s mRes at t 3d diffent dtie points using a serial dihdtn.of fourdifferent caiptureantibod concentrations 1ooM1 Figure 11shows therapy saiaing diagnostics and therapy montrng at various tnetnods ofte invntiocn aye usedI i nlence icha practice wih repect to s eetmng hoe appmeptate breast cancer herapy fh a particvdar patiatn 25 il*35]FinreIt how sngl cel snstivty detecting pHIERI and pER2 usig the {0061 Figure 12 shows the corrtSol of lEG and roximty assay results with lANA frmm frozen tisue [0037 Figure 13 (top) shAws a "heat map' illustrating the les of activted HERI. HIER2, HE5R3, P13K, SHiC and p9S fhrm FNA samplswith known H1ER2 IHC status. Figure 13 (bottom) shows a. Western blot analysis of totai HERZ2 and p 95 levels in a subset of the FNA samnpes with known HER2 M IC status, $ 0038] Figure 14 shows the conversion from W ER2~negative primary tumor (by ILIC) to HER2-positive CTCs (detected using the proximity assay described herein), j00 39} Figure 15 shows confirnadon of HER2 expression in CTCs by 10 imaging 00401 Figure 16 (top) shows the phosphorylated and toal HEIR2 revels in Herceptim t treated BL~T/R and BT474 eels. figure 16 (bottom) shows a W ebt ny o phosphorylated and total HER2 levels Ro .e2repdn-treatd BT/R and BT474 cella. {00414 Figure 17 shows that There was a&s.gnificant difkhrence in activated p95 levels between BT1474 and .BT/R cells and tht there was a redclution in phospho- HER3, P13K, and SHC in both BT 474 and BT/R clls with Hercepti treatment, iS 5 0f421 Figure t shows that there was an inhibition of phosphorvation of 1ER2 in BT474 cells with Hkrcepdn treatment. 100431 Fiaure 19 Yhows a schematic ofthe Ebth pathway in 7T4 ens in the abence of Hiercepin The inteasiw of the gravscxle it the tigore indcates the leel of activation (darker gray denoteshgher ihN! of ativahion 20 40944 Figure 20 shows a schematc of thm modulation of'the ErbB pathway in BT474 cells with Heepin treament, The intensitof the grayscaie in the figre indicates the level of ativati (darker gry desnes htgher level of activaion),. [(0451 Fgure 21 shows a schematic of the Erbm pathway in BT/R cells in the absence of 11ecepinTheintensity of the grayscale in the. Ugure inlae h lvlo civto dte 2$ amy denotes higher level of activation), 0046] Figure 22 shows a schematic of the :odulation of the ErbI pathway in BT/R cells with Herceptin treatment. The intensity of the grayscale in te figure indicates the level of activation (4rker gray denotes higher level of activationl. 100471 k'gitre 23 shon the anray design o exensary slide fIrnats f analying total 39 an phosphotylated HEPi and HO324(2 bvels.

!UQ4$*j .Figoar 24 shw cert fateeqavfQUbsai oxo HER2 j005 Figure 26 shows detection of total and aryaed 9t P19Q541 fgureV2 lsw detectio ofh toedan hoparyaet p tengthand enemed H1R in resstant but not sensitv celes. Cell ste wvas mnalyzred for expresson of otal and phosphcyated Ea )and fo totaE)and phosphorn(Aed. p95HERi~2 (D 0 0W*2] Figure2Thshows treotment wih Uemcepli ad level ofaactvaiton f~s nd K>n snsive BT474) and resistant (BT/R)ells at diferentime pedio 10-53 Figure 29 shows a schemato daxemplary pcorit assay fortaatorzewtenn taland phsphoreiav& . s.a gasiucer aiterest M*541 l~uire30 shows Che istr but v for aclivateHER? HUER2t expresedlER2 5 tMrR2x and the Icvdt of K J0Q5j gu3e 1 shows the CTC4IERstatus conversion o e 0o hc0 cohor The 1IRR2 status mynvsio isntat. foM &is PHbFt and tiRP. Tementi 20 10057] Fiure 3hows fnton l.HER profiling of CCs by COPIA ad FISH CI 10 ceas 0015Ij igure 34 hows p9511ER2 expression and activatierni primary breast eancet [*9591 Ftgure 35 shoWS Ihe correlAn bewee h tE2epesinsau dtni by 2$ EX e C Samples wth discordant HER2 stat a between the lIO and Ilmetm ethos are Tidenfed ir (006cq Figure 3 shows ann 4 sem analysis that was permed to conimthe HER2 axpressiontatuis in dincrdant sa ples. ampsesvish discordant 1E1R2 status teiweeu C and.CPA w<ere, tllthr vstpaed fbr theIU;2eprsio vf 'esen Asueto ~0~t IF~hra 7 how exmpls o fnctona pahwy prefiliing by (0 1 A, $ Example l' PW)632 Figmn~ 39 v' '~vds an ex amp of athwa profIlin on am EA-s'peob-, F64~ Figare 40 provides a econd p ate of pathway prodldb n o.3 FNA sampl ob frmI a patiettwidetastate breast cancer obanad frm a pader wth vmetsaic breast cancer. P066 Figure 42 poie a forth example ofpatbw p o anNA sape obtained fm a paent ith metastat breast cancer [0-0.71 Figure 43 provids (a) T evt exo of tR roand o2 an ENA seni e obtained front a patieMB468 aSKtri bresescey 10061 Fgur 44proidesa sxthcan.pe ofpatwayproilig o a F A sampt (pospoyin ed aHtERvi withretse cal can er.eiiTKatvto o ebwt o RTK69 expreo (RF poies (d seenohgractwee oferivhwd fmmce n awith vamgdere 20 ErbB-RT Fiuex4prsies MAnegt MB-23p1, M oA-f~ patha ngT44 on) anENA samples one2stgd fon na patient ofitER2,etC3+ breast cancer. @0711,. Pleurav 47! shows (a) The usactvton ofllt T and" -ui1ER 2 atasf~tvt ee fa r gle cellin, MA%t68 and SKr3 recivly (bs)Weerbndaageatdfo 2 4go oa ptin e lanJ (apoi mataely 4W1 cels) (a Th auii of cels reg'uird 2$ dtct! K sgnal wsatra:in 1o 1200(0. 1URFQ inHr R 1 'i iphosphorylated BE-71 o p.USR Ima 20Ane i toMl fzo.xan BCA $12,- -ofTNR2,-iUC 3t.- 7 of I4ERJ1C It 7 otHERI 1 1 1. and 4 nonnal adpacenats wote ardyzed trHR21-1ERI eXpression and ativador (t)Scater p 6of BCA saNle R ota HER2 -xpsion di o, 2 phosphorylariont 072-1 Yigure 48 shows: (a) For each siide, a standard curve consisdAg of serially diluted 5 cell sate was prepared fmhm states of ceo lines MD-46 (HER1positive) and SK r3 ( ER2-positv) cells; (b) Thel ofdetecnm (LD) value -was determined to be less than 1 CU fo btatNh pHER1 and pHlER2; () A total of 27 breast cancer samples amyyed fir their E ICexpression and activation are shown in the table, 74JU~ Frre provides an overview of a proessfor data reduction and data analy 1 R734 Figarc shows stanar enses and a cakcdatio for one ain.yte jO1)tj The figures and tabs fromPdCTPublieraionN WO20090863 ae heren nmpoaed by reffenoein heir etrty for all puose DTAIU% DiSCRIPTION OF THE INVNTRON 5: [ traction [00%iOR As described above. the activation of s gnal transduction pathways that tare invo Ived in cel prollrraion and he deaivation of pathways that are involved in eU death ae non miriting examples of molecular features that characterize many different types of cane in may e s moeulzar ga s for a given ype of cancer. Such acvtie mmpoin 20 my tsher provide usend targets for therapeutic imervend . Aecordingy knowl edge of the activity level of a particular signal transduction system within a cancer cell prior to. during, and altetreatment provides a physician with highly reevam formation tht may be used to select am appropriate course of treatment to adopt Furthermore, the continued nmonitonng of signal transduction pathways that are active in cancer cells as treatment 2-5 progresses can provide the physician with additional infonnation on the efticacy of t reatment promaptng the. physician to either continue a particular course of tsatmrent or to switch to another line of treatmemt. when, tort exmpe cancer cells have become resistanrt to tteatmenit through trfher aberrations that activate ecther the samea or another signal transduction 30 10171 Accordingly. the preset invention provides methods ad comupositroas for detecting. the exprersi ont and/or activadion, states of a plurality of deregtdated srgnal transducton 12 Mio s A umor in:t isSU orC xaotmrd cells such as rare circiadmng cells of a solid tumor ia specific, multiplex, .high-throughpuit assay. The i wvetionl also provides methods and compositions for the selection of appropriate therapy (single drugs or combinatious of rugs) to down-regulate or shut dove a deregulated signaling pathway. Thus, th invention may he 5 used to facilitate the design of personalized therapies for cancer natients, '8i The ability to detect and identify tumor cells in the circulation through the determination of rho activity of signal tranduction pathways at the level ofaingle cells is an important advantage of the present invention, fumior cells are ellen found in the blood oft patents with various early stages of caner as "microtastases (dissemnated tuamr cells) 0 and are also tnd in metastatie cancers. The number of turnor cells in blood will depend on the stagceand type of tumor, While biopsies are typically obtained on primary tumors, most metastatic tumors arc not biopsied, making molecular analysis of such tumor samples very difficult During tumoer mnetastasis, the moost aggressive tumor cells leave the primary tunmor and travel throngN the blood and lymphatic system to reach a distant location, TMa. Scirculating tumor cells fem blood represent the most aggressive and homogenous population of tumor celts, However, the amber of metastatie tumor cells in blood is frequently very low, varying fronm one to several thousand cells per milliliter of ood. The ability to isolate and assay signaO trsduction pathwaya in such rare cells nod to apply this information toward more etftoive cancer treatments is one object of the present invention 2(1 [00'791 In some embodiments, the muhiplex, highThroughput immunoassays of the present invention Ca detect the activation state of one or more signa transduction molecules in circulating cells of a solid tumor at the single coll level> in fact, signal transduction molecules such as EGRJI can be detected with a senstrvvty of abou 100 zeptomeles and a linear dynamic range of from about 100 zeptomeles to about 100 Ttomoles As such, singletceii detection of the activation state of multiple signal transducers in rate circulating cells facilitates cancer prognosis and diagnosis as well as the design of peonualized, targeted {TII8$O Rare circulating cells inchle circlatigells of a solidtmor that have either imetastaised or nucrometastasie frm a solidruroeCirculatings tumor cells>cancer stemn 30 cellsand ells, that are to grating to a tumor (g:, due to chemom tmetion) such as cclating endothei al progenitor cells, circlating endothelial cellsnirulatfog prts~mgtogce mycid Zvi 511 a Wdow1.3 clls nud circidatdng dendaiti cols are anae examples of citetdating celiassociated wit a $)$8} Signal transduction moicodes of interest arc typically extracted shortly afoer the ciuing cells are isolated to Preserve their in si3U activtion state, peferably within about j~" n A' -- -~ c-n' tf'i'4 anerbt tyl 24 6, or i ra m about 30, 1 . r miutes.T isolated cels may also he icubated with one or nore growth f usually at maomair to microuoiar cocerationts, for about I-30 minutes to resuscitate or stimulate activation of the signal transduction molecules (ye.g, Irish at al Cell, I18217-228 (2004)), 1 OO82f As explained ra potential anticancer therapies tor 10 an individual patient, the isolated cc cant be ineated with one or more anticancer drugs at vYring dosv Gth factor stimulation can then be performed for a tew mnt( g about S minutes) or for several hours (og, about 146 hours. The differential activation of signaig pathways with and without anticancer drus can aid in the selection of suitable cancer thetvpy at the proper dose each individual patent Cimuting cells can also he 15 isolated orn a patent rample doing anticancer drug treantci and stimulated with cne or more growth factors to dtermi'nen whether a change in therapy should be imtpiemrented. As such, the metods of the presnt invention advantageouslyasis the clinician in providing the "ih anticancer drug at the right dose at the right time for every patieri 10083 Wi h regard to breast cancer, current testig options are unsatisfactory because 20 tranent of both primary and rnetastic tumors in a breast cancer patient is hased on a one time diagnosis flrm a biopsy sampe taken during an catly stage of the disease, in particular, terapetic intervention for both tre early and metastatic stages of breast cancer is based solely on the initial diagnosis from the biopsy sample taken during an carly stage ofthe disease because of the impracticality of obtaining a biopsy sample Tom a metastatic cancer 25 patient However, breast tumors are evolving as a function of time and treatment such tha temporal moitorng of breast rumors is crcai for optimal Imnaement of breast cancer patients, For example, a change in the activation state of ore or more of the EbB (tER) famnity of receptor tyrosine kinases masy affect therapy selection at rorefnenc, inideed, discordance in HER2 status between prinmary and nmetastatic cancer is common because up to 30 37% of allarest cancer patients change hrm a HER2-negative primary tumor to HER2 positive metastatic cancer in addition, patients may have denovo resistanc or develop acquired resistance to hornonal therapy due to HERI/2 activation. a some instances, pat-ients may have de. nmve resistance or develop acquired resistance to ErbB-targeted theapies due to the presence of tunor cells expressing p95HER2 As a result, there is an uet clInical need to assays to assist the clinician in prescribing the appropriate cancer therapy at the appropriate time bame current technology lacks sensitvity and specificity, cannot be used to monitor patients on therapy, and do not utilize pathway profiling to guide individuaized teatreent decisions. {041 In contrast to currently av ajiabe breast cancer testing options the methods of the present innt enable the monitoring otf breast cancer patients through all stages of the i proving areal-time biopsy" of solid breast tumors using samples such aS ci arculating turmor cells (CT Cs) fhrm blood and/or fine needle aspirates (FiNAs). As a non nating example. the breast cancer assays described herein can be used in the initial diagnosis of breast cance in a patient at an early stage of the disease, Selection of a suitable cancer therapy is guided by prowling theexrsson and/or atdvation states of' specific SignaliNg pathways with and without anticancer drugs using the assays described herein. 5 Advantageosw the methods of the present inveion can also be used to monitor the progression or regreswon of the disease because herape-utic i.terventinn may be based on samples taken at any atage of the disease and analyzed using the assays described herein A s such, selection of suitable excer therapies tar the early and tetastatir stages of breast cancer is gusided by reaildime diagnosis and an analysis of the expression and/ot activation status of 20 specific sigrnaling pathway molecules. [00Q85] The methods of the present invention are beneficially tailored to address keys issues canton u~nd pro, Wideafo in. cancer management an'roiea higher standard of care for breast cancer patients because they (1) provide increased sensitivity (eg:, mle celf detection cant. be achieved for detecting total and phosph<ylated signal transduction molecules such as EOFR and PlER2i 25 W~ oath~ hncrdse 'p-''' r 2 ,z(2 provide increase pe ifiit4(. treceatibody p rity asasenhnnce specificity for detecting total and phosphorylated signal transduction moleculess. () enable pathway profiling (e.g. express ion andior acti sation snatus of specific signal trasduction molecules can be deteered in CTCs or F-NA Thon patients and (4) eliminate any issues with obtaining patiet samples (eg. assays cant be prb edon a few tumor cel) Ahhoug any sample 30 may be used in the assays described herein, CT C are partiiuly uasefu because they represent the most aggressive tumor cells, Cyvry tumor is known to shed CTCs, they can be the only source of residual tumors or hardIonccess metastatic tumors, and they ate found in blood. As such, the methods of the present invention enable the serial sampling of breast ti5 tnmor tissues, resulting invaluble informatieru on changes occumar in tumor ce 8s as a imneton of imaesdrherapy andproviding cdincians with anrs to tvnioraprdi evoluan cAncir patd wyinnatre 008 n sun the omnpoitions and methods 0 f the pres ern i Ien an adagerdi provide acurate pedictin selectic Mand monitoring o cancer patts (ag mast ancr padSeS sgmost I Kely to benef fron tageted therapyby peonning patha pig n eaiy accessible tumor cens using atiol ed add ased prontoty ssays. I0O7 s used herein de ng ha the meanings ascribed, to thenn unleL Il specified otherwiser 10088) The term "cancer- is intended to include any member of a cass of diseases characterized tUdgrowth of aberrant cells. The term inudes all known cancers adneoplastle conditions, whether characterized as mtalignant, benign, soft tissue, or solidand chance of all stages and grades including pre- and post-etastatc cancers. 1$ REapes of diffrent types of cancer include, but are not limited to, breast cacer; lumg canCer (eg, non-smafl el bung can cert; digestive and gstrOitestinl cancers such as colorectal cancer, gastointesnal stroumal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, ble duct cancer, small intestine cancer, and stomach (gastic) cancer; esophageal cner; galbladder cancer; liver cancer; pancreatic cancer: 201 appendix cancer; ovarian cancer; renal cancer (ag , renal cell carcinromaV cattcer of he ce tral nrvou system; skin cancer: lymiphomas ehorincarcinomnas; head and neck cancers: osteogenic sarcoma: and blood cancers, As used herein, a "tumor" comprises one or iore cancerous cells, In one embodiment, the breast tumor is derived from a subject with an inv"aive or in situ form of'ductal carcinoma or lobula~r careinoma,. In another emrbodtiment< 25 the breast unmor is derived from a subject with recurred or metastatic breast cancer. {89J The term "analytc" includes any molecule of interest, typicaiy a msacromnolecule such as a poly peptide, whose presence, amount (expression level), activation state, and/or identity is determined, In ce r amaces, the analyte is a signal transduction molecule such as ag, a components of a HE2 (ErbB2) signaling pathway, S0{O 90 The tern "signal transductionr molecule" or "signmal mr sducer"includos proteins andi otber molecules that carry out the process by which a cell converts anr extraceilular signal :16 roa respon typkally involving ordered scquoes ot bioChemiCal twzt'sm inside te cel.. Exaples of signal trnsdioim mickules Vnclude, hut are not l imited to, receptor tyrosine kinases such as EOFR (eg, EOFRCHERIl . HER2/NerbB2, HER/ErbBt3, HER4/rB V C4), VE R 1./FilI. V EGP'R2/FLK!KDR. KFR3/L W4, S PLT3/LK2, PDGFR (eg., PDg FRA,W DFRB), c-KITSCFR, INSR (inulin.recpt IGF4R. lGPF-lKR, IRR (insuli rceptor-related receptor CSF R54, PfGRR 1~4, HGF R P-2, CCK4, IRK A-C. c~MTT RON fEPHA P. EPHB 16 AXL, MER, TYRO3, TIE 1-2, TEK R YK , DDE. 1<2, RET, &-ROS, V-cadhein, .TK (leixoyit tyrosine kinase), A LK (anapiastic lymphoma Uinase), ROER I2, MUSK, AATY( 1.3, and RTK 106; trncated 10 fAnns of receptor tyrosine kinase such as truncated .HER receptors withmiing amino termiuna extraczlular domnains (a . p95Erobi2 (p95m),. p11!0, p95c, p93n, eWa); receptor yosine kOnae dimar (eg p95HiAR2tI/HR3, p91HER2/HER2 HER2/HER2, HER2!HER HER I /11ER2, HER2/HER3, HFR/HER4, et): non-receptor tyro ine kinases such as 3CR A$BLSreeP FrBik, Cak Abi, Zap70. Pes/Fps, ia. .Iak, Aok, and LI MK: tyrosine kinase 1 signaling ,ascade components such as AKT (eg AT I K'12 KT3 MElK (MAP2KO)> ERK2 (MAPK I), ERK. (MAPK3), PISK (aeg- P1K3CA (p1.10), PIK3RI (p8S)), PDKi, PDK, phosphame. and trsbn hnnlg (PTFN), SGK3, 4 V71P, P70g6K (eg p? S6 kinase slice ovarian lpha 1), protein tyrosin. phosphaoaes (yg, PIT B, PT'PN 3B W.), RAWF PLA2, M EKK W TN KK, JNK. p3K Shl (p66) R as (e ., K-Ras, N-Ras, H- R) 20 Rho, Re l. Cde42, PLC, PKC, p53. cyIn DI, STA T1, ST A3,n hosphatidylinositol 4,5 bisphosphate (PPL phosphatidyinoaitoI 3A4 tisphosphate (FP'3), mTOR.. BAD, p21I p 2 7 , RUCK W 3 TSP -1, NOS, GSK341, RSK 1-1 INK c-dun, Rb, CREB, Ki67, and paxilli; nuclear hormone receptors such as estrogen receptor (P progeskrone receptor (PR), androgen receptor, glucorcoreid receptor, minerahtorzowli receptor, vitamin A 25 receptor, vitamrin. D receptor, retinoid receptor, rhyroid hormone receptor, nd orphn receptors; nXUckW rocepto coacivators arnd pessos such as ampified in breast oacer (AIB I) and nuclear receptor corepressor 1 (NOR), respectively; and combinations theof (009tj *The term componentt of a H-ER2? signaling pathw ay" inchides any one or more of an upstream ligand of HER2, binding parner of HERT2, and/or downstream effector molecule 3( that s miodulated through H~p ER2 Eamples of HER? signaling patluvay components include, but are not limited to, heregulitn HE E/r'b~.. HEW:2! ErbB2, HIER3/ErbB33. HER4/Eirb4, AKT (Qg., AKI, AKTZAKTS). MEK (MAP2K1 ERK2 (MAPK), ERK (MAPK3), P13K (rg, PIK3CA (p 10), PIK3RI &p8S) PDK I, PDK2 PTEN . SOKT, 4E-BP1, P706K 17 (cg, wPH variant alpha . proxn tyro ine phosphatass (:g0 PTPIB PT 3 EDP st RER dimers (eQH pHER2ER3, p3iER2/HER2;VER2/H.I2 .HER2'HER3, jiBRto/HEER2"Rrs tER3 WLR2HER4awl GSKiiAP112, PTiESp27,and cttmioadns 5 [0092) The tem "tivation state" refers to whether a paricular signal trasducnan molecule suob as a 1HER.2 Signlang pathway comlponenft i activated, *Simaialy, the term "A leve" refer to what extent aprtuar signal tranaduction moeule such as a HER2 signahlg pathway component is activated, The activation state typically corresponds to tire phoporyvation, ubiquinatin and/or~ complexation status of one or' moresgd tO transduction molecules, Noritimng examples of activation states (listed in parentheses) include: EGFR (EGiFRviii, phosphorlted (p- EFR, EGFR:She, ubiquitinated fu~ EGFR p-EGFRvUi); ErbB32 (p-Eb2, p95HER2C (trunca trbB2l p-n9iHER2 ErbB2S he, Er bB:iK ErbB2:IEC FR Eb2-:ErbB3, ErbB2:EIrbB34): ErbEP (p-RThB3. ErbB3 :P13K, p ErbB3PIK, SrW3:She); ErbB4 (pxbB4, ErbB4:She); c-MET (p+MET); AT (p 15 AKT); AKT2 (p-AKT2); AKT3 (p-AKT3); FTEN (PTFN); P7056K (p~P7O86K); K (p-MEK); E RK1 (p-ERK1); ERK2 (p-ERK2); LPDK1 (p-PDK1); PDK2 (p-PD); SGK3 (p SGK'C); 4E-BP I (p-4E-BPi); PK3RT (p-PIK3R1); c-KIT (pKIT); ER (p-BR); IGF1 R (p IR IG i-R:IR'l TRS:PK3, p-RS, KF-1 R:PK); INSR (pNSR; , T3, (p-FLI' T3) HOFRI1 FHGF 1 0FR2 (p-CGFR) RET (p-ET) PDGFRA (pPDGFR A); PDGFRE 20 (pPDOFRB); VEGFRI (p-VEGFRL VEGFRI:PLCy, VEGFR :Sre); VEGFR2Q(p VEGFFR2 VEGFER2:PLCy, VEOFR R2Sre, VEGPR2:beparin sulphate, VECOFRI2:VE~ adhering); V EGFR3 (p-VEGER3); FGFRI (p~FGFRI); EFR (p-PGPR2): F R (p FFR3); FGFR (p-FGR41; TIM (p-TEl); TIE2 (p-TI2) PH2A (p.-PP A) PEB (p.

EP : GSK-3 (p-SK-35; NFKB (p-NFKBK -&KBpP65:IS) BAD(p-AD, 21 1BAD;14-3-3A mTOR (p-mTOR); Rtk-1 (p .k-I); Jak (p~lnk); P3 (p PV); STAT3 (p STAT3); Fak p-Fak); Rb (p-Rb) 167; pS3 (p-p53); CREB (p-CREB); c-Jur (p-c-nY); Src (p CSte); ad pax0li (p-paldn [00931 As used herein, thec term "diluaion serhes" is intended to include a series of decndn concenrati> of a particular sample (agt cell iysate) or reagent (eg aruibody). oA dilation series is typically produced by a process of mixing a m redtamount of a sa concentntion of a sample or reagent with a dilucnt (eg, dilution buflr) to create a lower concentratio of the sample or reagets and repeating the process enough times to obtain the desired number of serial dilutions. The sample or reagent can be serially dilated at 1 s eai244 5 6, 7, 8, 9, 1$0, 0 1 5, 20, 2550, 10 500 or 1.0old to produce a diionseres comnpsig ateast 2,3, 45,6, 7,g, 940. 11, Q2 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 descending conetrations of the sample or reagent, F-or ,a dtion series comprising a 2~Ald serial dilution of a capture antiboy xeaget at 5a 1 mg/mI starting concentration can be produced by mox ing an amount of the starting conceentration af capture antibody with an equad amrovnt of a dilution buffer to create a 0.5 mg2/ml concentration of the capture antibody and repeationg the process to obtain capture antibody contcentrations of 0.25 mg/ml, 0,125 mg/mL. 0,0625 mg/nmd, f025 nS g/m, e& I*O94) The te suprior dynamic rane" as used herein vees to the ability of an assay to detect a specific anyte in as few as one cell or in as many as thousands of cels For example, the immunoassays described herein possess superior dyon range because they advantageousiy detect a particular signal transduction molecule of interest in about 1-10,000 cells (eag, abomt I, ', 10,.25, .. 0, 7$. 100s, 250. l00, 750, .1000, 2500, 5000. 7500, or 10A000 cells) using a diution. series of capture atbd ocnrtos 10,095 As used herein, the terra "crcultrng cells" com prices extratumora cells that have either metastasized or micrcmntstasized from a solid tumor. Examples ofccrlating cells include, but are not limited to, circutatig mmor cells, cancer stem cells, and/or cells that are migrating to the tumor (eg circulating endothehal progemtor cells, circulating endothelial cels, circulating prouagiogenic ryeloid cella circulating dMndritic els. en,), Patient 20 samples containing circulating cell can be obtained from any accessible biological fluid (e.g, whole blood. serum. plasma, stun, brancha 4 tavage f uine, nipple aspirate, lymph. saltva, fine neredle aspi rate, et.), in rertajn instances, thre whole blood sample is separated into a plasma or srum fraction and a cellular fraction (it. cell pellet). The cellular fraction typically contaios red bood cells, white blood cells, and/or circulating cells of a solid 2t tumoer such as circulating tumor cellis (CTC4) circulating endothelial eils (CEC4) circulating enadcthelial progenitor cells (CE/PU), cancer stemn cells (CS~s), disseminated tumor cells of the lymph node, and comnbinations thereof, The plasma or' serum faraton usually contains inter' ala, nucleic acids (eg. DNA, RNA) and proteins that are released by circulating cells of a solid tumor, 30 ~tM1906 Circulating eIs atypicallyisolated fromi a patient sample using one or more separatin mahods including, for eampl, mmnomagnetic separaton e eg.l Rila e s-7Po Acad S A SA, 9:4569494 (1998); ilkemotb e a n A 19 92:37-52 (001), te Cllnaks" '' Sy tb Im nmicon (IHuntngdon Valley, PA), microthmdic separation. (see, e.g, Mohamed et ar, IREtE rars. Nanobiosct, .3:251i-256S (2004; in at at, Abstract No. $14?, 97th AACR( Annual Meeting, WashintonDjC (20 C (Fee Aeg, Mancuso eat a BWood, 97:3658-661 (2001.) density gradient $ eentrifug~ation (see, eg, Baker e at, Oin Cmeer Res 1348548-71 (2003)), and depletion methods (Pse. Moye e a' h /n. Oncob 21:521-530 (2002)), {97] h term "ampl as used herein includes any biological specimen obtained nom a patient :Sampltes includ~evA wit 13tOhwout iittow oibood, plsa erm e blood cells, Me Waool 1. nnr -white blood cenls (e., peripheral blood msononuclear celast ductal aaefuitnpl 10 aspirate, lyrph (eg, disseminated tumor cells of The lymph node), bone marrow aspirat, saliva, urine, stool (ia, fees), sputum, broncdaI lavage fluid tears, tine needle aspirate (e0g> harrested by random pertasreolar fine needle aspiration), any other bodily fluid. a tissue sample (e., tumor tissue) such as a biopsy of a tumor (enxg needle biopsy) or a lymph mode (eg. sentinel trymph node biopsy), and cellar extracts there ofl In somec emibodlimenus the 1 sample la whole blood or a fractional component thereof such as plasma, 5 serum, or a cell pellet in preterred embodiments, the sample is obtained by isolating circulating celis of- a solid tumor from whole blood or a nelluiar fractlion thereof usmng any technique known in the art In other embodimecnts, te samnple is a formasli nfxedi paraffin embedded (FPE) tumor tissue sample> etg, frnm a sold tumor of the: breast 20 tO098 A biopsyy" refers to the process of removing a tissue sample for diagnosttce or p3gnosla evaluation, and to the tissue specimen itself, Any biopsy technique known int the art can be applied, to the methods and compositions of the present invention. The biopsy technique applied will generally depend on the tissue type to be evaluated and the size and type oflthe turner (lte. solid, or suspended (I. e blood or ascitesj), among other factors, 2.5 Representativ biopsy techniques include excisional biopsy, incisional biopsy, needle biopsay (g core needle biopsy, rine-sneedle aspiration biopsy, etc.), surgical biopsy. and bone muanow biopsy. Biopsy techiues arc: dismissed, fot example, hrrrrsan i- Piiphrn of .thrwnai Medicine, Kasper, at at, edt, i6th ed., 2005, Chapter 70), and trout Part V, One skilled in the art will appreciate that biopsy techniques ca be performed to ideatity 34) cancerous and/or precaneerous cells in a given tissue sample. "' qA nAw I"Psy "' [099 The term subjecd ovpaten or i individuaFy includesumans but an as nie othe& imal~ stii a5 other prnnate r detS itl .atiehnes. e4Unin ne5a pcrcnesnd dhe like. U0100 An "arry" or miroray"' comprises a distinct set and/ox dilution series of capture 5 antibodies irnuoized or restrained on a solid support such as for enmuple, glass (em, a glass slide). plastic sachip 0 pins, filters, beads (egag beads poystyrene beads et1 paper, mrnbrne (a~g o, nc r ~ocelltose, poiyvinyak dene fluoride (PVDF etc.), fibI bundrles, or any other suitables substrate. The capture antibodies are generaxlly itmmbtzed or .restrined on the solid support via covaem or nonCOVakl intactions e IOnic bonds, 0 hydrophobic interactions, hydrogen bonds an der Waats forces. dipola-ipole bonds), in certain instance~, the capture anti bodies comprise capture tags which intract with capture agents bound to dhe solid support The arrays used in the asays described herein typically comprise a plurafny of different capture antibodie'suad/or capture antibotdy conucentrations that are coupled to the surface of a solid support in different known/addiressabie locations. O] The term "cap ture antibody" is inended to include an immobilized antibody which is spcifi for (o. binds, is bound by, or fors a complex wilt) one or more analytes of interest in a sale such as a cellular extract In particular embodiments, the capture antibody is resuained on a sond support in an array, Suitable capture antibodies for iaobilizing any of a variety of signal transduction molecules on a solid support are 20 available &om Upstate ( Temecula, CA), Cell Signaling Technoiogies (Danvers, MA)M I systems (Minneapoh, MN)> Lab Vision (Femont CA) Santa Crus Bjtechnoldogy (Santa Cruz, CA), Sigmna (St, L ouis, MOL~ and BD1 Bioscienceis (Sa os CA {()1121 The ean "dctwon antibody" as used herein includes an amibody comriss ag A 2. detet ahe labei whiA js specific for (t. binds, is bound by. or frms a complex ydt) one or nme analytes of interest in a sample. The term also eoompasses an antibody wich is specific for one or more analytes of interest, wherein the antibody can be bound by another species that comprises eectableiabet. nmpics of detectable Yabels include, but are not ited to, blotinstreptavidin labels, nucleic acid (eg oigonucleotide) labels, chemically 20 reactive labels, fluorescent abes, enzyme iaioeis, radioactive labels, and combioaions thereof. Suitable detection antibodies for detecting the activation state and/r total amount Cf any of a variety of signal transduction molecules axe available from Upstate (Temecua CA> Biosoure (Camanlilo. CA, Cell Signain~g 'iechnologies (Danvers, MA), R&D Systems (Minneapolis, MN), Lab Vision (Fremont CA) ama Cox Blotechnology (Santa Cruz, C A) Sigma (St, Loais, MO , and BD Bioscens (San Jose CA), As a non-limiting examp phoph-spciic ntbodesagans various phos phorylaied fes ofsga eduction mofllectes suh as BCGFR. JT, c~$c, FLKl PDGFRA, PDGFRB, AKT, NAK, PTEN, Raf. and MEK are available from Santa Cruz Biotechuology, 101 O3] The term "activation statedependent antibody" incides a detection antibody which 'is specit for ( e, 'binds, is bound by, or torm a complex with) a particular activation state of one or more analytes of interest in a sample in 'preferred embodiments, the action 10 state dependent antibody detets the phosphorylation, ubiquitination, and/or comrplexation state of one or more analyes such as one or more signal ninnction; mrolecules. In some embodiments, the phosphoryiadon of t member of the EGF'R faly of receptor tysione kinases atduor the 'formation of heterodimeric complexes between £i(FR faxmily members is detected using activation state-dependent antibodies, in particular embodimnents, activation iS statedependera antibodies are usefuP for detecting one or more sies of phospborylatior in one or more of the following signal transduction molecules (phosphorylaton Wsits correspond to te position of the aino acid in the human protein sequence): EGFR/HRI/ErbBI (e~g, tyos (Y) 1048); ErbfB2flER2 (eg Y1248); ErbB3/H 'ER3 (2 A- I E 2 ErbP4 /E414 (ag, Y284); SGK3 (eg, threoni ( 256 and/or serne (S) 422' 4-OP (e.g. T70); 20 ERK (eg, T202 and/or Y204); ERK2 (e , ME'K S2' , 2 andor S22M) 1%3cR (e g, Y688); 'PDK1 ( g., s24); P706K e, T229, T389, anda S421); -M*EI (e'g, Y1349); PTEN eAg, S380; AK '1 (, g, S475 and/or T308); AKT2 (eog. 5474 and/o 1309), AKT3 (e8g. 5472 and/or T"S); (SK-3j (eg. M9y N FKB (e S36g. $530 r IKB g832 BAD ( Ug, S112 and/or S3) mTOR ( 524b; R6sk) (eg, T357 and/or 3) a k 25 (eg T83 and/or YI85) P38 (e T180 and2/or V182); TAT3 (og Y705 and/or '727); FAK (g. Y576 Rb (emg, S249,T252, adr70 p53(eg, 5392 andor S20); CREBl (eg, S133); c-Jun (a.S63); c~Sac (&g., Y416) and paxillin Ig Y11), 1010| Te tem "cdvdan tat-indpenentantibody" includes a detection antibody which is specific 'for (L , binds, is bound by, or fbrms a complex with) one or more analyses 30 of interest in a sample irrespective of their activadon state, For exampic, the activa'ton state independent antibody can detect both phosphorylated and unphosphoryhtetd frrms ofone or more AalyS tes sh as one or mor signal rans'duction molecules, 22 [O105) The term iucleic acid" or "p olyrucleotkid' includes deoxyribunelectides or ononucleotides and polymers thereof in either single- or double-stranded form such as, aTy example, DNA and RNA Nucleic acids include nuclei acids comaining known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occuncing, S and non-naturaliy occurring, and which have Wimilar binding properties as the terence nuclei acid, Eixamnples of such analogs include, without limitation, phosphorothioates, phosphoramidates methyl phosphonates, c mtl. phosphonates, 2'-methyi ribsonucleotides, and peptide-nucleic acids (PNA4). Unless specifically limited, the turm encropsses nuciete acids containing known analoguresof natural nucieotides that have 10 siilar binding properties as the reference nucleic acd, Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompassea conservatively modifted variants thereof and complementary sequences as well as the sequence explicitly indicated. (0106) Theaterm "oligonocleotide" refers to a single-stranded oligomrer or polymer of RNA DNA, RNADNA hybrid, and/or a mdietic thereof, in certain instances, oligonucleotides are 15 composed of naturally-occurrinig (L r. unmnoditd) ncleobases, sugars, and internucienside (backbone) linkages in certain other insumnces, ol igoinucleotides comprise modified nucleobases, sugars, and/or internucleoside linkages. |W107I As used herein, the tern "mismatch motif' or ^'mismnatnh region" refers to a portion of aneognucleotide that. does not have 100% complemnentarity to its complementary 20 sequence, An oligonucleotide nmay have at least one, two, three, four, five, sixm or more mismatch regions. The mismatch reg ions may' be contiguous or rmay he separated by 1, 2 4, 5$ 7, 8h 9, l10, iiL 12, or moore nucleotides, The mismatch motifs or regions may comipri se a single nucleotide or may compise two, three, four, five, or mnore nucleotides. h)008 The phrase "stringent .hybridization conditions'' reters to conditions under which an 25I oligonucleotide will hybridize to its comspleme~ntary seq uentce, but to no other seq uences, Stringent conditions are sequence-de perndent and will be diifferent in. different circumstances, Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nuclei acids is found in Tijasen, Techhns in Blinchemitr and Moleculamr Bfiology-Hybridizaran with Nucleic Probes. "Overview of principles of hybridization and 39the strategy of nucleic acid assays" (1 993), Generally, stringent conditions are selected to be about 5-10rt lower than the thermal melting point (.1) for the specific sequence at a defined ionie strength pH-i The TI, is the temperature (under defined ionic strength, pH, and nucleic nb2 coneraton) at which 50% of the probes complemAentary to the target hybridize to the target sequence at equilibrnriu (as the taget sequences are presentmi excess, at T14 50% of the pObes ae occupied at equilibrium), Stingent condiions may also bc achieved with the addition af& demahvin sgents such as formnamide. For seltetive or specific bybridimauon, a positive signal is at ieast two times backround, preferably 0 times background hybridization, 10109] The terms "sutan'tially identical" or "sustantiai identityQ in the context of' two or more nucleic acids, refer to two or rore sequenrcs or sbsequseoces that are the same or have a spcified percentage of nucientides that are the same (Let, at least about 60%, preferably at 1.0 least about 65%, 70%, '75%, 80%, 85%, 90%, or 95% identity over a specified region) when compared and :ligned for mamum correspondence over a comparison window or designated region as measured using a sequence comparison algorithm or by manual abgvnrn and visual inspection. This definition when the context dictates, also refes analogously to the comrpilemem of a sequence. Prcferaby, the srhubsmtiai idetntvt exists over a region that is at least about 5, 10, 15. 20, 25. 30, 35, 40, 45., 50, 75, or I00 nueleotides i length. I9a T he term inceubatiag" is used synonym~ously with 'Tcontacting" 'nd exposrngand des not mply any speelieimne or terture requri'rets uiess otherwise %dicated. H DOescripO-,, af' th audintd 20 jo1fl I The present invention provides conptonsr andmethod for deetc the status (e. express and/or activation levt1s) of components of signal transduction pathways in trmor cejs derived fron tumor tissue or circulating cells of a soli tmor wvith a assay s.uch asa specdic, multieM high-throughput proximay assay as dscribed herein. The present invention also provides compositions sad methods Ibr selectintg appropriate therapies to 25 downregmlate or shut down one or more deregulated signal transduction pathways. Thus certain embodiments of the invention may be used to facilitate the design of personabzed therapies based. on the partien lar molecular signature provided by the collection o~f total and activated signal transdoactin proteins in a aiven patient' s tumor. 101121 In particular aspects, the present invention provides molecular markers (biomarkers) 30 that enable the deterinatioo or predtion of whether a particular cancer can respond or is likely to respond favorably to a iERt2'-modulating compound (e.g, a HER2 inhibitor). t pecifie embodiments, measuring the level of activation of one or mone componts of te 24 MER2 sigading pathway (es. HER2, p905HER2, hiR3, PGK p9SRER2/HERI HERf. and/r SHC) is particularly useMb fo determining or predicting the sens itivty of cels such as breast Oancer cels (eg isolated circulating tumor celS= fe ee aspirate (FNA) cells> and the like) to corpounds that mndulate ER2 activity (e monocloal antibohes such as tastunuraob (Hercen'ptin) tyrosine kinase ihibitorsk and the ike), f1113| In one aspect, the resent invenon provides a method for determining or predicting the sensitivity of a cell to a compound that modulates HIER2 activity, the method comprising: (a) contacting the cell with the comipoumci; (b) losing the cell to produce a celhdar etact; to (N) determining the expression and/or activation (sg, phosphorylation) Ieve of one or more components era H4ER2 signaling pathway in the ceihdar extracA; and (d) comparing the expression andor activation level of the one or more components of the N ER2 signaling pathway determined in step () to a Reference expression andi/or activt*ion level of the one or more compponet of the An FR2 signaling path wherein a diffuerce between the expression and/or activation level of the one or more components of the 1 ER2( signaling pathway determined in step (ct) and the reference expression and/or activat on kevel of the one or more components of the H:ER2 signainmg 20 pathway indiates that the ccii is sensitive or msistant (L a., not senstve) to the compound 0*1 14 in referred aspects the present .twention provides a methodafor detern ng or comiprising ()cntacting the cell with tromnpound; 2$ (b) ing the d wp a celiue xtact; a) deteIr iting the activationlevel ofLHER2 or p9$FR2 n te celoa extract and (d) etupading the activation, level of flER2 or p95H1ER2 determined in step (a) to a reference actiaion level ofUHER2 or p95HER whereinthe pence of a ghe e of HER2 or pb5HER2 activaonin te c.elidar extract ormard to the reference zusdwation leve of 1ER2 ore5OR2 indicate that the cel isnot senive resistant) to the compound 2$ (0115 To preserve the in air activation states, sia induction proteine such as HER2 pathway components are typically extracted shorty after the ceils are isolated> preebiy within 96, 72, 48,24, 6, or 1 brt more preferably within 30, 1S> or 5 minutes. The isolated cells may also be incubated with growth fctors usually at nanomolar to nicrotolar .5concentrations tor about l-30 minutes to resuscitaite or stimulate signal transducer activatica see. g Irish a at, C , 1 R8:247-228 (2004) im story growth factots ilude epidermal growth fatter (EF), heregulin (HRC), TGF-as PGF, angiopoietin (Ang), NRG PGF. TNF-a, VEO. PDP1GF110, RF., HGF, cytokimes and tie le, To evaluate the sensitivity of the isolated cells to a compound that mnodulates ER2 activity (e., an anti 0C HER2 monoclonal antibsody uoh as trasuumab) the isolated cells can be iacubated with the compound at varying doses prior to, during, and/or after growth factor stimulation. Growth &ctor stimulation can be perfbrmed for a few minutes or tours (eg. abou 5 minutes to about 1-6 hurst After isolatioru treatment with the HER2-modulating compound, and/or growth factr stiNuktion, the cells are tysed to extract the signal transduction proteins such I. a HER2 pathway coponents using any technique known in the art. PreferaTy, the cell yisis initiated between about 1~260 nxutes alter growth factor stimuliation, and more preferably at two different time intervals: (I) at about 1-S minutes after growth the-tor stiulatiou; anid (2) between about 30-180 minutes after griowthi factor siimuliation. XMternatively, the lysate can be sted at 40"C: until use, 20 (41 I6) Nor 4imiting examples of comnpoundsi that modulate 1HR2 activity include mnonoclanal an>todies tyrouine kinase inhibitors, tad coumintons thereof. In preferred emboimentsl thme HIER2.modamaing compound inhibits HER2 activity and/or blocks HER2Z signaling, a g. is a HER2 inhibitor. Examples of ER2 inhibitors include, but are not hmited to, monoclonal antibodies such as trastuzumab (Hiercepti) and pertusrruah (2 (4); small 25 moleeul-e tyrosne vinase inhibitors such as gefitiib iressa"), erltinb (reva" pilitnb, CP-654577, CP-724714,* canertinib (C.I 1033). .HK1272h lapatixib (0W-57207 6; Tyikerb%. P10d66, AEE788, BM.-599626, }i-357, BiBW 2992, ARRt334543, JNJ-26483327, and JNJ26483 327; and corn biadions thereoE in other embodimemi, the HERT-modulating compound activates the .HER2 pathway, e~g, is a uJER2 activator, 30 [0117] In soee e-mbodments the reference expression. or activation level of one or more components of the HLR2 sgang pathway (e.gz, HAER2, p95HRER2, yHER3, P13K, p9SHER2/HER3. HER I SHC, etcj is obtained from a celi sensitive to the compound (eg, a He-rceptm"~ -senstive cell) that in treated with the compound (eg Heeptin"), in certain 26 embodimnen~ts, the cel sensitive to the cOrnpOund (L et comapound-sensitive cell is selected fro th grup consistin of a BT1-474 cell, SLBR3 cell, NH27 cell, MDA4MB-361 e ell U.ACC412 cellt UACC493 cell, MDA-MB3-175 cell, SUMI190 cell. SUM225 cellt Nf7 cell. OYEl9 cell, and combinations thereof. Sac, e . Tseng er at, Ml Pharmarc oI 70:1534~4 (2@006); Waiberg er ;:l. Clin Cancer Res, 1 6:1509-19 (21); Emnlett a, ot. Ca ncr 7her.. 6:2664-74 (20~07); Koneeny et aL Cancer Ries., 66:1630-V9 (200 6 t In somc instances, thecomoun-se~dtve elllaengineted fromn an existing celi or cell line (eag a compound resistant cell or cell line) to create a cell or cell line that is sensitive to the compound (eag. by expressing a 1-ER2 signal~v~ ingpahay compoxn (e g ER2?) miodulated by the compound 10 in the cell or cell line). Preferably, the comnpound-senesive celi is a Hierceptnsenstive cell such as a £T474 cell. t018 lai other embodiments, thre reference exprsasion 'or actvation level of one or more components of the HER2 signaling pathway (ag, HER2, p95HER2t, HER3,% P13K, p95MER2/RE3R3, HER I.. SHC, etc.) is obtained t'rome a cell resistant to the compound (a, a C5 OM~ -75 SJM O 1-kHeeptn resistant ceii) tha is treated with the compound (eog, He~rceptin), yIn certain emnbodiments. the c-elI that is resistanat to the conmpound (La e ompond-resistant cell) is selecte:d from the group consisting 'of a $T/R cell MDA-MB~23 I cell SKBR3I/IGFlR cellt JIMT, I cell BT474/HR20 cell, SKBR3/P2 celt NU29- ell NUi47 cell, MCP'- celi, MCP 7/71 3 celi, MCF-.7/HER2A1 6 cell ZR-75I celL, BT120 cell, M-DA-MB~433 cell, 1471 ce!l 20 MvDA4MB-453 cellt MDA-MB-468 cell. CAMAI1 cell. MDA-MB-157 cell. PPM I92A celi, KiLl celL, EM19 celi, C~AL51 cell NUOO3 cell, NUC4 ccli t 1U97 cell, SNUI ( celL and comisaticus thereof, See, crg. Tseng et al. Mo. .PharmncoL 70:153-4 (206; aiberg ea, C/hn Cancer Res. I I6: 1509-19 (2010): Emlet et al. Alo/s Cancer T her- 6:2(664-74 (2007); Ktmeeny at aL, Cancer Res. 66:1630-9 (2006) in some instances the compound ~ resistant cell ia engine-red from an exsting cell or cel linc (eig a co Ioun-sensitive cell or cell line) to create a cell or cell line that is resistant to the compound (vg b y knockirig crut a HER2 signaling pathway components (eag. Hi-ER2) modulated by the conmpond in the cell or cell line). Preferably, (he cornpound-resista cell is a Hlercepti& resistant cell such as a HT/R cel. 30 [1: 19] in further embodimens.he derence -xposs ivon egel gf one n or toe coruponems of the HER712 sgnaling pathwayje ( HER2, p95E~R2. HER13, P13K, p9oO 5NE/iEtK E1KI SHC. ae is obtained from a cell such as a tumor cell that is not tHted with the conpound(g, erceptint a some instancesthe tumor cell. a hhea UAI()0~ o?, A cancer cell obtained from a patient sample, in other instances, the tunor cell is a gastric can cell obtained from a patient sample, i further finances, the tumor cl is a .metstatic tmmfor cell whOSe primary Origin is either o breat or gastriC cancer cell. in ptict un embodiments. the cell that is not treated with the compolmd is obtained from the same sample 5 that the isolated cell t~g, a test cell to be interrogated) used to produce the cellular extract is oibtunied. [012tii in certain embhodimnents, a higher level of expression: or aehivatiof ra ETR R2 signaling pathway conmponenit (e~g. HE2 95E2 HE3. 3,p5ER.E3 H1311, SHiC etc) is considered to be present in a cellular extract when the expression or 10 activation level is at least about 115 ,25 0 3.,4, 4,5 5,5,6,65 7 7 , 59, 9,5, 10, 15, 20, 25, 30)5, 40 5, 50, or 100Ot)ld higher (eg about 1,5-3, 23 2-4, 2-5, 2-l10 2 20, 2-50, 3-5, 3-1 0,3-20,3-50 4-, 4-10 4% 4~50 5-10, 5-15, 5-0 or 5-50-fld highr) than the reference expression or activation level of the corresponrdirg IR2sinaling pathway component in a compound-sensive cell (eg. BT4-474 cell) rated with the 15compoun, in a compound-resistant celi (a. g., BTR cell) treted with thie comoporud, or in a cell (e5g a tumor cell such as a breast cancer cell, a gastric cancer cell, or a HER2-expressing tuorcell obtainedirom a patientsample)not mrited wish the compound. 0121 In other embodim ents, a lower level of expression or activation of a HE pathway component (ag. HER2, p95HEfR2, 11ER 3, P 3K, p95 ER2/ER3 HER.1, HC, 20 etc:) iS con stdered to be resent in a cellular extract when the expression or activation Level is at least about L52, 2,5, 3,L,4,4.5, 1j5, S, 6,3,7, 75, 8 859, 9, 10, 15,202 3Q 35, 40, 4i 50, or 100old lower (eg, about 1 5-3, 2-, 14, 2-10 2-20, 2-50, 35, 3-10, 3-20, 35,4-4-10 4-2t 4,50, 50, -15, 5-20, nr 5-50-fold ower) than the referee expression or activation level of the corresponding HER2 moling pathway component in a 25 compoun-sensitivc cci (e:gO 1-474 cell) treated with the ompound, in a compound resistant cell (e.g, BT/R cell) treated with the compound, or in a cell (e g., a tmor cell sAch a\ a breast cancer celi, a gastric cancer cel, a a HER12-expressing unor cell obtained from a patient sample) not treated with the compound, [01221 iroume embodinents, the pr'ence of a higherlevel of ER2 orp95E2 30 actl atlorn in theellulartacnt comrpated to the rfersnen activationlevel o 1ER12 or p95H2in a campoundsensiive cell indicates tacell e themes cell from which the ceiltlarextact was prodnoed) isnotsensitive estanto the compound I other 'NO5 c dimenits.the presence of a similar or lower level of hER2 or p95HER2 activation in the cellular extract compared to the reference activation leve of HER2 or p95 ER2 in a coudsensitive cell indicates that the cel (.gy th rest cell from which the ceihilar extract was produced) is sen sitivec (5 e. not resisttat) to the compond, in one enbodimemr 5 the level, of HER 2 aAivation in the cellular extract is at least 2 to 3-fold higher thn the reference activation level of ER2 in acompound-sensitive celi (ag, BT-474cel In another embodiment, the weSl of p95 TER2 activation in the cellular extract is at least 5-od higher than the reterenc aictvaion level of p95IER2 in a comtpoundhsenstuve cell te&, 'l 474 cell 10 WP1231 In some embodiments he presence of a lower level ofF1ER2 or p95HERZ acivaon in the cellular extract compared to the reference activation level of HER2 or p9HRR2 in a compound-resistzut ccll indicates that the eel) (eg the test el trom which the celIula a mexat was produced) is sensitive (Sce. not re embodiments, the presence of a similar or higher level ofHER2 or p95RER2 activation in the 1 cellSar oreact compared to the reference activaton level of HER2 or p9JIER2 in a compoundresistan cell Lidicates that the cell (eg. the test cell from wich the celhdar exmTact was produced)is not sensitive Se reistan) to the compo [.0124j in some embodiments, the presence of a lower level ofHER2 or p5 t ER2 a~ctivationin the cellular eXtract compared to the reference aci vanon level of HR2 o 20 p95HER2 in a cell (e.g, a tumor cell such as a breast cancer cell a gastric cancer cell or a HER2-expressing tumor cell obtain from a patient sample) not treated with the compOnd indicates that the cell (mg, the test cell frm which the celldar extract was produced) i k e, not resistant) to the compunl. In other embodiments, the presence of a similar mr higher level of H1ER2 or p95HwR2 activation in the cellular extract compared to 25 the rarnce activanon level of HELR2 or p95HER2 in a l n ated with the compound indicates tat the cell (g the test cell fm which the cellular extract was produced) is not sensitive (he. resistan) to the comipoamd, j{U25} i hcedain enmbodiments, the method conttprises detenmninig the activation level of both HER2 an p9HER2 in the celluarextract Inparticar embodimnthe aedvaton 0 tevel of RER2 or p95HER2cornpriscs agplwaphatloe leve of HER2 or p95iEtiQ 0126 n cein other emimntni the methd :ltbr comprises etenntig the activati.on leve of oe re additional ignal rasduction molecules in the cellular 29 extrnet, Nonlimiting examplies of additional sigala transduct ion molecules inchde EGFR (HER1) HER3, HER4, P3K, AK T IIMEK, PTEN, SGK3, 4EP ERK2 (MAPKin ERK1 (MAPK3), PDKI, I,0S6K, GSK-3p, SHC, IGF~R, -M T, c~KiT, VEFR 1 VEGFR2 VEG(FR(3,receptor dimers (a g. p95H1ER2/fER3 hetarodimner, HER2/HER2 homodimer, H ER2/HER3 hetewdimer, HER '/HER2 heterodimer, and/orilHER2IHER3 heterodimer> and combinations thereofi In particular embodliments, the activtion levels 4ofl one orK~ moetfthe add itional signal transduction mnolecues comprise phospihorylation levels of such mnolecules, In 1 itrtrabodimenats, the mnethod comprises determninirtg the activation level ofCHER2 and/or p95HER2 in combination ith one or more of HER3, P13K, aud/or p95HER2/HE3 10 heteroditner in. the celludar extract 101271 In some embodiments, the method further or aleratively comprises determiing thei acti'vation level of one or more of HI3R3, PD3K. and/or p93HE2R2/RiER3 hetorodime. In certain instances, the presence of a higher level of EER3, PI3K. or p95iE;R2/HER3 beterodimr activatiron in the: cellular extract compared to the reference activation level of 15HER3, P1DK, or p93HER2/HER{3 heterodioser in a comnpound-sensive cell indicates that the cell (etg, the test cell from which the cellular extract was prodned) is not sensitive (i ., resistant) to the compound. in other instances, the presence of a similar or lower level of' HIiR3, P13K, or p9SIER2/lIER3 heterodimra acivfation in the cellular extract compared to the reference actvation level of HER3 PDK, or p93HERZ{ER3 he der in: a 2C compoundsensidve ccll indcats thattecell (g ', t est cell from which the cellar extract was produced) is sensitive (it;, not. resstant) to the comipond, in one em bodiment, the level of HER3 activation in the cellular extract is at least 2 to 34old higher than the reterace activation level of H.R3 in a compoumnd-sensitive ell (e.s, BT-474 cell). [0128] In certain lnstanc, the presence of a lower level of HER3, P13K, or 25p95.IER2/UlER3 heteradimer activaion in thet cellular extract compared to the reference activation level of HER3, PI3K, or p95HEiR2/RER3 heterodjimer in a cornpound-resistant cell indicates that the cell (eag, the test cell from which the cellular extract was produced) is sensitive (i e, not resistanrt) to the comapound, In ether instances, the presence of a imlar or higher level ofHER3, P131K or p95HER2/HER3 beterodimer activation in the cellular extract 30 compared to the reference activation level of TER3, P13K, or p95HER2/HER3 heterodimer n a cormpoundresistant cell indicates that the cl (e the test cell from which the cellular extract was pro'ducd) is not sensitive (he. resistant) to the compound.

[01291 In some istaces, the presence of>a lower level of REFR3. P13K, or p95l4ER2/iJER3 heterodimer activation in the celular extract compared to Uhe refrence activaton level of 13ERA 3,PK r p R2ER hetarodimer in a cPl3 (eLg. a tumnr cell such a a breast cancer cell, a gastric cancer cell, or a ER2-expressing tumor cell obtained S from a patient sample) not treated with the compound indicates that the cell (C g, the test cell fmin which the cellular extract was produced)l is sensitive (i a' not resistant) to the compound In other instances, the presnce of a similar or hwghr level of HR3, P13K or p95.HER2/ R3 heterodimer activation in the cellular exttet compared to the reference actriation level of' HER3, P13K, or p9HR2HI heterodimer- in a ell not treated. with the 10 compound indicates th ththe cell (0g, the test cell fmm whici the celular extract was produced) is not sensitive (ie, resistant to the cmpournd, 6130 som embodiments, the cell tag, the rest cell from which a cellar extract is produced) is a tumor cell such as a breast cancer cell, a gastni cancer Cell, and/or a E-R2 expressing tumor cell. In certain instances, the tumor cell, is a circulating, tumor cell or a fine 1$needle aspirate (FN A) cell obtained from a tumor, in other embodiments, the cell (e~g, the test cell tom which a colinler extract is produced) is isolated fro a sample that is obtained. e~g.. hum a abreast or gastric cancer patient, Non-limiting examples of' samples include bodily fluid samples such as. for examuple, a whole blood, serum, plasma, ductal lavage iluld, lpple aspirate, lymph. bone marrow aspirate, urine, saliva, and/or lme needle aspirate (NA) 20 sample. In particular embodiments, the sample comprises a whole blood, serum, plasma, and/or mmor tissue sample such as breast or gastric tumor tissue or iTER2-expressing mor tissue I#1M 31in certain instances, the methoed may further comprise the step (e) of providing the resut of-the comparison obtained in step (d) to a user (ng, a clinician such as an oncologist 25 or a general practitioner in a readable format, in somc instance 4 the o may fluther comprise enAdmg or reportmg the result of the comparison obtained in step (d) to a clinician, e ., an onuotogist or a general practitioner, ln other instances, the method may further conmpriserecording or storing the result of the comparison obtained in step (d) in a computer database or other suitable machine or device for storing information, eag, at a iabortory, 30 10132 in some embodiments, determining the -activatinevel of one ormore HER2 signing pathay components (agt HtER, p95dHER2 ERZ, PK131 p95HfER/HfER3 HERI ad/or 8HW)in step (c) comnprises detecting a pho-aphotylation level ofe-ne or nore HER2 signing pathway components in the cellular eartract with antibodies specific for the phosphoryla ted form of each of the H-ER2 sngrabng pathway componars to be detected, P11331 Phosphorylation levels and/or status can be determined aing any of a variety of teczhniques. For example, it is well known in the art that phosphorylated proteins can be 5 detected via mmunoassnys using antibodies that specifically recognize the pho phylated form of the protein (se ag Lin a . t a I, . Cianar, 9313721381 (2005)). hmuinoaays generally include immunoblotting (eg. Westem blotting), A, and EL A. More spec types of niunoassays include antigen capture/a-ntigen competition, amibody captre/atigen competCitiontwoi ibd s wicea antibody capture/antibody exess, and antibody ID captumre/atigen excess. Methods of' making antibodies are descrbed herein and in H-arlow and Lane, Antibodies: A Laboratory ManuL I 98$, Cold Spring Harbor Labontory Press, Cold Spring Harbor, NY, USA, Pho~shpecif antibodies can be made de novo or obtained rwm comomercial or noncommnercti sources .Phosphorylation levels and/or status can also be determined by metabolicaliy labeling cells with radioactive phosphate in the ferm of t P] TPp wl or {yP.ATP. ylated protein become radioactive and hence traceable and quranti liable through sintiaion counting, ra-diogniphy. and the lika (see, e g, Wang c at BloL Chem 253:27605-7608 (197$)), f/or example, mnetabolically labeled proteins cano be extractod f-rm c&lls, separated by gel ectrophoresis, transfebrred to a mxemubrane, probedl wit an antibody specific fhr a particular HER2 signaling pathway component and objected 21 to autoradiography to detect P or OP, Alternatively, the gel can be subjected to autoradiograph prior to member transference and antibody promgn, 01 34] in particular embrxliments, the activation (gg, phosphoryfation) level and/or stats of one a nore i ER2 signaling pathway onponens (eg, HER2, p95HER2, HIE 3 Pf3K .p5H 2 /HER3, HER, and/or S1) in step (c) is detected with an immtmoassay such as a 2 sngl deectonassy o a roxniy dual dletection assay (a a a CQlaborative Proximmoity 2 5 Ani eeto assay or ai avi ImunoAsasay (COPiAA)) as described. herein, 101351 In certa embodiments, detenining the activation ( .. , pho herylato,) level o one or moe opponents ofa HE 2 ignalng pathay (el, HERL HER2 IER3.t i. step (cornprnse% 30 (1) inculnting (a contadig) the ceharxtractwith a diItion series of capture antibodies (a cature antibodies spec W e rHER2) to fcan a plurAd ity of captured waytes (a g. captured receptors, wherein he capture antibodies are 32 restrained on a sold support (eg to trnsform the anvayte present in the clular extract into complexes of capturedi analytes coprig the analytes and capture antibodies); (ii) incubating (age otactmg) the pl urality of catured analytes (ex, captured 5 receptors) with detection antibodies comprising activation state-dependent antibodies specific fohr the corresponding anailytes (eg activation state dependent antibodies specific for HERZ2) to form a plurality of detectable capttured analytes (eg. detectable captured receptors) (ea., to trea~ns the complexes of captured analysts into complexes of detectable captured analytes I I) comprising the captured analytes and activation state-dependrent antibodies); (iii) incubating (esr, contacting) the pluraliy of deteale capowred analytes (ang, detectable captured receptors) wit firt and sNcondTh members of a signal amplification pair to generate an amuplitied signal; and (iv) detecting the amplified signal rented from the first and second members of the signal amplification Pa 1t1161 hInertain other emtbodimnt5 deterniang the acovtin (aphosphnrylation) level of one or more t'unatedireceprora of aEHR2 signalig pathway tug. p951lRt) in tep( comprises; (i) incubating (g~ contacting) the cellular eettract with a phety of beads specific th an.xtracallular domain (ECD) bnding region of a fiaifeagth receptor (e, i idcingth 1TER2): (i)remuving th phaiyo ted rom thle celula e~xtact, thereby removing thC e thidlength receptor (e~g.. fPUbenath. HER2) to form a cellular extract devoid of the fulklength. receptor (ae. fulklength. H ER2) (e&g, to transform the cellular 2. extract into a cellar extract devoid of a specific fuit length receptor or faunily of fitlength receptor) (iii) incubating (eg contacting) the cellular extract Kdevid of the fhtlengt receptor (e~ full ngth RER2) with a dijlution series of captue antibodies specific for an intt3elluar domain (ICD) binding region of the fulblength 30 ~ receptor (ag. full-length HER2) to form a plurality of captured truncated receptors. wersin the capture antibodies are restrained on a solid support (e, to trnsmformn the truncated receptor present in a fiulaiength receptor-depleted cellular extract into complexes of tuncated receptors and capture antibodies); 'V3 (iv) inuating (e~g., contracting the pisraliiy of captredtrtd receptors with detection antbdies omprsingi activation state-dependent anttiies specitRt for an ICD binding region of the fullength eceptor (eg, Auilength HERC) 0 form a phuraiity of detectable captured trnicated receptors (:, to transform the complexes of captured tn cated receptors ino compjee of detectablfe captured truncated recptors comparing the captured trunrcated recepor anid activation are-dtependttr antiodies) (v) Wcubatng (eg:. comactn) te plurality of detectable captured truncated receptors whh first and second memnbenrao a signal apiification pair to 10 gene a a amplified siga: and (vi) detecting an amplified signal generated from the first and second members of the signaampliication pair 01371 in sonme instances, the activadon state-dependent antibodies comprise a irst member of a binding pair (eg, biin). ha other tinstances, the firsi member of thesignad 1$ amopli feation pair (ecg., a peroxidase such as HRP?) comprises a second member of the binding pair (eg streptavid t certain instances, dhe second member of the g amPpification pair may be for example, a tyramide reagent ( g, bioin-tyranride), Preferably, the ap'i'ed ignal is generated b peroxidase oxidization ofhiotin-yramride to produce a activated tyraide ( g, to trnsfon the bitin-tyramide into an activated 201 tyaidel 'The ativrated. ratmide nay be directly detected or indirectly detecte, eg, upon the addition of a sigamdltctinurg reagent, Non-iitig exiamples of signal-detecting reagemts include streptavidindlabeled flunorophores and cominaetiom of'atreptavidin -elted peroxidases and chromugenic reagecnts such as. ag, 3.3'5tetranethyibenridine( TMB), 101I38) The truncated receptor is typically aAfragmaent of the ftdllength receptor ando shares S ain b rg regon with the full-enegth receptor, in certain embodiments, the fidl-enth receptor comprises an extracedhular dovrmil(CD)binding region, a transmemb rane domain, and an intracellular domain ((C) handing region. Without being bound to any particular theory, the tAncated recepto ay arise through the proteoiytic processing of the EC.D of the full-length receptor or by alterative initiadtn of translation 30 fronm methionine residues that are located before, withmt, or after the transmuembrane domaint, eag: to create a truncated receptosr with a shortened ECDf or a truncazcd receptor comporisng a membrane-associated or cytosolic fICD (tagmtent. 20 a~mAut si 0I1391 In, cnin prefered aembodim ents, the tnmcated receptor is P95HE12 and the corresponding fofliengthi receptor is HIER2. ilowever, one skilled in the art will appmecate that the methods described herea for detecting truncated proteins can he applied to a num of dinhernt proteins inning but not limited to, the ECFR VIlI nmtant (implicated in glioblastoma. colorectal cancer, cx), other trinated receptor tyrosiez inasos, caspass, and the like, Exampje 12 of PCT Publication No, WO2O R&9/10 37, the disclosure of wich is heret inrporated by refrenace in its r irety for all purposes, provides an exemtplary e mbodient of the assay methods of the present invention for detecting truncated receptors such as p95HER in cells using a multiple. high-tbrmghput, single detection mirorray 10 ELISA having superior dynamic nmge. {014O] in some emrbodhlnehithe phuralty of' beadspecific tfo anEBtD Winding region comprises a reptavidiibiotinpair, wherein the optavidin is attached to the bead and the b0oi atached an antibody. certain instances he antibody is specific for the ECD binding region of the LUd1iengit receptor (.g. fulblength HER2) 15 [01411 in some embodiments. each dilution series of' capture antibodies comprises a series oftdescending capture antibody concentrations. in certain i stances, the capture antibodies are serialy diluted at least fold g, 2, S, 10, 20 50, M0, 501, or 1000-fld) to produce a dilution series comprising aset number (e.g. 2, 34. 5, 6 7, 8, 9, 10, 15,20, 25, or fiO) of defending capture antibdy concentratio which are spotted oto an array, Preferably, at 20 least 2,3 4. 5., or 6 ccepieatec of each capture antibody dilmiton are spotted onto the array. [01421 In other Imbodiments, the solid support comprises glass (eag a glass slide), plastic, chips, ins iters, beads, paper, mnembrane t eagronyonn nitrcelluo po linlde fluoride (PVD etc.), her bundles, or ay other suitable substate. In a peeled emuboditmenlt, the capture antibodies are restrained (e,.,. via covalent or noncovalent 5 interaction) on glass slides costed with a nitrocelulose polymer such as, for example, EAST Slides, which are commercially avail e om Whatnan Inc. (Florham Pak N), 01431 In certain embodimenrts, detennining the activation (e g. posphoryation) level of one or more componetrus of a HE.R2 signaling pathway (e4, HER1, HER2. HEi tc )w in step (c) comprises: )incubating (. contactIn the elluda extract wit a ditn series of capture antibodies e capture atibodies speifc for HER2) to tnn apluraity of captured maaytes (e~ captured receptors'twherein the capture antibodies tare 35 mestmined on a solid support (> g:, to transfo rm the analytes present in the elular ex act into complexes of captured analytes comprisig than and captre antibodies (ii) incubating (0g1, contacting) the pluaity of captured aalytes (eg, captured a receptors) with detection antibodies comprising activation tate-independent antibodies specific for the cornespondig anapytes ( g , independent atbdies specife fo HR) and activation state-dependent antibodies specific for the corresponding analytes (eg:, activation state dependent antibodies specific for HElR2)' to or a pluntdity of detectable 10 captured analytes ( detectable captund receptors) (etg, to transform the complexes of captured analyses into complexes of detectable c.tured analytes comprising the captured analytes and detection antibodies) wherein the activation sate-tdependent antibodies are labeled with a fciliating muoiety, the activation state-dependent antibodies are labeled with a first nmb r of a signal amplification pait and hailiat monety Senerates an, oxidizing agent which channels to and reacts with the first member o the signal amplfication pair; iag (e.g. containing) the plurality o detectable captured anaytes (e g detectable captured receptors) with a second mebe oftthe signal 20cramp lifiesttin pair to generate arn amplined signal; arid (iv) detectig he amuplitied signa! generated from the first and second meters of the signal amplification pair, 01l44] Itt (crtaint other arnbadimnentgyderetnn ithe activation (ag phosphoryhation) vel of one or more n cated receptors of a Isignag pathw a p95HLl2)in 25 step (ncompcarises: ) iubating (ag contact) the eular extraec wh a plurality of beads specificfo ar extraceflulrdoraitnEChD) binding rae of ah~as rneptor ge iRg. mIrnoving thea plrality of beads frozm the ediar extrectahereby ranaotng te 3 flength receptor { a.nibdlength EP11) o thnn a cellula tract devoid of the id~enh receptnr ( thlklengtUER2 to tmasform the cellular tract into a CeOlMar extract devoid of a specifit-length receptor or fanrly of fulenginreccptorsj 36 (iii inuang~ (eg4. comauctingi the celluar exthact devoid of the fuilength receptor (erg. fulklength HJER2) with an purality of capture antibodies specific for an intraclular domain (ICD) binding I re n of the fullength receptor (eg, fl~ength H-ER2) to form a plurahity of captured truncatedreptr wherein the capare antibodies are restained on a solid support (eg. to transform the truncatcd receptors present in afllegth receptor-depleted cellular etact into complexes of truncated receptors and capture antibodies); (iv) incubating (e~g contacting) the phurality of captured trusrcated receptors with detection antibodies comnprising activation state-independent antibodies and It) activation state-depenident antibodies s'pecific for an lCD bhinhg region of the Mrlngth recepror (ag, ful~ength HER2) to fo. a plhrality of detectablte captured tr'uncated eeptors (an, to tnsfr hecnplexes of' captured trncated receptors io complexes of detetable captured truncated receptors comririug the capwred iuncated receptors and detetion antibodiest 1$ 'wherein the activation stateIndepund cat antibodies are labeled with a faciltamting moietyw, the actition s pendent aid ath a first member of a signal amplifncation pair, and the facilitating moiety genewrates an oxidi rng agent which channels to and, reacts with the first member of' the signal amplikalimpani; 20 v)inad omatng) t i of etecte captured truncated receptors with a second member of the signs amplifiation pair to gene-rate an amplified signal; and vi detecting the anpified signal genated fomethaand d membs of the signal amplification pair 25 [45] The activation state-independet amibodies may he direl y labeled wit the facilitating moiety or indirectly labeled with the facilitating moiery, rg, via hybridiration between an oligonucleo tide conjuigated to the activation state-indeedent antibodies andi a try oigonu conjugated to dhe fWINing m ty Siily, th activation state-dependent antibod-ies may be directly labeled with the Trst member of the 30signal amplification pair or id irectly labeled with the first member of the signal amplification pair, eg, via binding between a first member of a binding pair corougated to the activation state-dependent antibodies and a second emuer of the binding pair conjugated to the first member of the signal amplification pair. In certin instances, the first member of the binding pair i biotin and the second o metber of the bindirg pair is an aidisucb as sipavidn fl 0 Lnta d tP146] in some embodimcats, the facilitating moiety may be, for Cxampk, glucos oxidase intain instances, the glucose oxidase and the activation statesindepenident antibodies can be cctjugated to a sulihydsy-ctivated dextra.nmolecule as described iA g, samples s 16 17 of PCT Publicatiom Na WO2009/108637, the disclosure of which is herein incorporated by reference in its entirety for al purposes. The sulhydsylacivtend dextran molecule typIcaiiy has a molecular weight of about l500ka (eg abot 25000, 350, 400, 450, 500, 51600 62t 7')0 550 600 650 ' 4 00, or 750kDa), in other embodiments, thet oxidizing agent may be, for tO example, hydrogen peroxide (H 0s), tn yet other embhodiments, the first mem~nlber of the signal anplification pair may be, for example, a peroxidase such as horseradish pemxidase (MRP), In further eiment, the second member of the signAl amplification poiray be. fbr examnpie, a tyramide reagent (0g, bioin-tyramnide) Prefraibly the amplified signal is generated by peroxidase oxidation of bioTin framide to produce an activated tyranide (*g, to transform the biotaraide into an activated tyramide). The activated tyranide may be directly detected or indirectly detected ag, upon the addition of a signal~detecting reagent Non dimnitmg examples of signaldetecting reagents include streptavidin-Wbeled fluorophores and combinations of streptavidindabeled prorxideses and chromogenic reagents such as ag 3,3 SS-tetramethyibenzdine (fMB), [(5 fi 47J In certain instances, the horseradish peroxidse and the acttersaon statedependent sattbodee can be conjugated to a sufhvyhayivated dextraatn moeue. The sudhhydry act Ated dextxan mAdecUe typiehly baa amolecuar weight .ao .a a 6 tout40l 45, 50 5S. 6 5, 70, 0 8 5,90 95 or 100k a). [14t] The truncated receptor is typically a frag ment ofthe fuillength receptor and shares 25 n intracellular domn (CD) binding region with the fuli-length reeptor. In certain rmbodimns., the fhllength receptor comprises an extracelunar domain (ECD) binding region, a trnsmembranve domain and arn intracelldar domain (LCD) binding region. Without being bound to any particular theory, the tncated receptor may arse through the proteolytic procesig of the )CD of tO full length receptor or by ahenative initiation of translation 30 from mnethionine residues ibat are located b rwt.or after the inmsmembranC domain, a g. to create a treated receptor with a shortened ECD or a irucated receptor comprising a mrembraneassociated or cytosolic CD fragment, 101491 in certain preferred embodiments, the runcated receptor is p95HER2 and the corresponding fulenglh rocceptor is UIERt2. However, one skilled in the art wilt appzeciate that the methods described lhereinifor deteting irnaled proteins tan be applied to a n>umber of different pteins including, but not limited to, the EGFR ViU mutant (implicated in gliblastoma, colorectal cancer, etl) other Inmtesd reeptor y ine kinses, caspases, and the like, Thttnmple: 12 f> PCT Pablicatoion oVW2009/108637, the disclosure of which is herein incmpoated by reference in is entirelytot al purposes, provides an exemplary embodiment of the assay methods of the present invention for detecting truncated receptors such as p9$tlER2 in cells using a mhilex, high-throughput, proximity dual detection 0 mieroarray ELISA having superior dynamic range, 101501 in some embodiments the phuality of beads specific for an ECD] bindingregon. comprises a streptavidin-biotin pair, wherein the strepacyidin is etched to the head and the biorin is attached to an antibody. in certain instances, the antibody is specific for ite ECD binding region of the ful-ength receptor (e.g, ful"ength ER2), 5 [0151] In some embodiments, each dilution series of capture antibodies comprises a series of descending capture antibody concentrtions, hi certain instances, the capture antibodies are serially dilated at least 2-old (e.g, 2, S, I 0. SO, 100, 500, or 10old) to produce a dilution series comprising a set number (eg 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 2.0 25, or more) of dtescnig capr anibd cocnrtin hc ar spottednan aray. Preferably, at 20 least 2, 4, 5, or 6 replicates of each capture antibody dilation are spotted onto the array, 19152j ia other embodiments, the soid support comprises glass (eg, a glass slide, plastic chips, pins, filters, beads, paper, metbrane (ae.. nylon, nhrroceilose, polyvinylidene fluoride (PVDki), ec timber bundles, or any other suitable substnte. In a preferred enmbodiment, the capture antibodies are. restrained (e.g. ia covalent or nonenvadent 25 interations) n glass slides coated with a nitro celluose polymer such as, fo example, FAST'*Slides, which are comimercially available from Whatman Iac, (biorhamn Park, NJ) 5 ll another aspect the present neitn proidcs a method. predicting the response of a tumo to acompoud that moduates HER2 activity he method c ang contacting a cell obtained them he tmor with the compound; (0 (bllysing the cell to produce a elluar extract 39 (c) determining the exprsion and/or aciain(g phosphoryltion) level of one or more components ofn aI2ER2 signaling pathway in~ the cellular exract; andt (d) comparing the expression and/or activation level of the one or more cor~ponents of the HER2, signaling pathway determined in step Vc) to a reference epession nd/or activation level o:F the one or more components of th HIER2 signaling pathwy whceri a difference between the expression and/nr activation level of the one or more components of the HER2 signaling pathway determined in step (o) and the reference 10 expression and/or activation level ofthe one or more components of the HER212 signaling pathway indicates thet the tumor s or is not likely to respond to the compound (eg;, the tumor has an increased or decreased likelihood of response to the compound), f54a In preferred aspects, the present invention povides a method for predicting the of a tmor to a compound that modutdes lER2. activity, th method coprisng: 5 (a) contacting a cell obtained frorn the tumor wI h the cenopound: (b) lys ing the cell to produce a celhdar extract; (c) determining the activation levei of 1HER2 or p95HEiR2 in the celbalar extract; and (d) compalng te activation levetlof tHER2 oD 9$ER2 detertaed in step 0 (e) to a' refereccation level tiHER2 or p95HE2R2, whereintwhe presence of a higher leave f HER/12 o p95R2activation in these celarextmot comparedtthe melrence act nation level of WER r p951-1R dicat that the tuWmr s not likely to respond muihe compoundgg the tunor hasd ielihood of response to the compound 25 P155] Non%4mitng examples of compounds that modulate HER2 activity include mnonoclonali antibodies, tyrosine krinase inhibitors,. and combinations thereof' in preferred embodients the HER2-miodtflatinig compound inhibits HER2) atiity and/or blocks HER2I signaling, e.g is a HER2 inhibitor. Examples of'HER2 inhbtor iude, bunt are not limited to, morocional anibodies such as tmasmtzunmab (Herceptin) and pertuzumab( 30 moecle tyrosine kinase inhibitors ach as getitinib (fesa erotinib (Tarcevat) piiit CP-654577, CP-724714, cnertinib (CI 1033), KAM272, iapauti (GW-5720G/ Tykerbh 5 PKi-16n6, AEE788, BMS-599626, HI-3 57, BIBW 2992, ARRA -4543, iN3-26483327. 40 end ?Nl 264 83327; and cofi aatons thereof ia other embodimuents, tie i(R2&iodlating cornonud ivdtes fihe HEW ip~ailnvays g, is a 1HP2 actliator 1561 in some rebdiments, the reference expression or activation level of one or mote cponents of the HER2 signding pathway (a 1 , H1ER2, p951HER2. HER3, PIRK 5 p9$HER2/HEBR3, HERI SC etc.) is obta ied from a cl sensitive to the compound (eg, a Hceptin eniiUve ccl) that i tHceptinI n certain oo ~ ~ ~ snv TOya VQaCOB) is sected embodimemts the cell sensitive to the comipound (i a'. comupotmd-seniiecl) ssle troml the group COnlsisting9 of a BT-.474 cell, SKBR3 cellt NH270 ceit MDA4MB-361 cell, UACC12 celtUACC-93 cell, MDA-MB 75 celt UM 190 cel SUM225 cell, N87 celk 1 E19 Cell and comnbiadon thereof, In some instances, the compound-ensitve cell is engineered from an existing cell or cell line (eg a pnrsistant cel or ell line) to create a cell or cell line that is sensitive to the compound (a'g., by xssig a H ER2 signaling pathway component (e ., HER2) modulated by the compound in the cell RC cell lineY Preferablv the compound-sensitive cell is a Herceptifl esitive cell such as a 11474 15 cel [o157 in other embodiments, the reference expression or actiation level of one or more components of the HER2 signaling pathway (eg, RER2, p95H1ER2, HER3, P13K, p95E.R2/HER3, HER. tHC, etc.) is obtained from a cel resistan to the compound (tg. a HNcseptn~esistant cell) that is treated with the compmmd (eg,, Herceptin), In certain 20 embodiments, tie cell that is resistant to the compound (.e un d-resistam cell) is selected from the group consisting of a BT cell MDA~MB-231 cell SKBR3AGF RW cellt JIT-I cell, T-474/R2V col, SKBR3/P2 cell h29 ceL NH47 celt MCP- cell, MC 7/713 call MCF,7/HER2 6 red /R-75- cell. £BT20 cell MD AR.W435 cell, T47Dt cell. MDAMWB453 cell, M DMA -MB-46 ell. CAMAI cellt MD-B- 57 cell, *M192A cell. 25 KPL 1 celt FM19 cell, CALS ell NUGC3 Mo, L.L NUG4 Aelt FU97 cell SN 6 !,elL and combinations thereof. in soe instances, the com "ound-resistant cell is engineered from an existing cell or cc line (e.g a compounsenbsitave cell or cell line) to crate a cell or cell line that is resistant to the compound (ag, by knocking out a HER2 signaling pathway component (e> HM) modulated by the compound in the celd or ceU line), Preferably, the 30 compcsmd-rssistant cell is a feaceptin* resistant cell such as a BT/R cell [0158) in further embodiments, the refrence expression or activation eveli of one or more comrpoiets of the IER2 signaling pathway (eg, HER2, p91HER2, HER3, P13K, 41 p95HER2!HER3, HEM. STC, eo) is obtained from a cell (etg, a tumor cell such as a breast cancer cell, a gastric cancer cell, or a HER2PZ-exprsing tumor cel obtained from a pte sampfr) that is not treated with the compound (aE g, erceparn). In rtar odiments, the cell that is not treated wVth the compound is ontatted from the same sample that the > soiated cell (ag, a test cell to he interrogatd) used to produce the cetlluar extract is ohtained, 101 59| In certain embodiments, a higher level of expression oir activationi of a HER2 signaling pathway component (eg, HER2 p95HiER2, HR3, PI3K, p95.HER2HER3, HEZRI, SHCO, e,) is consIdered to be present in a cellular extract when the expreson or 10 activatn level is at least about I 5, 2, 2.5, 3, 5, 4, 45, 5, 5,5, 6, a.5, 7, 7 , 8,83, 9, 9. 10 20, 25, 30, 35,40, 4550, or 100.-fold hiher (e., abou 153, 24 2-5, 2 0 2 20 ' 2-0, 3~5, 3-10, 3-20l, 3-50, 4-5, 4-10, 4450 S~-A0, -15,S 5-20, or 5-50O.-ld higher) than the reference expression or activation level of the corresponding HER2 sig nhlng pathway composner n i a convpound-scnsitve cell (eg BT7-474 cell) treated with the compound, in a compound-resistant cell (EPg, BT/R celI) trded with the compound, or in a cell (erat a tumor ecl such as a breast cancer eall t ' gstric. cancer cell, or a thR pressing tumor let a btained Iom a patient sample) not tremed0witt the comrpomd. [016001 In other embodimrents, a lo wer level of expression or activation of a HER2k. sinaling pathway component (e., QHER, p9511R2, 4E2R3, P13 p9HRHE3HERI, S11C 2Q ae) is consideredto be present na euLar extt when tc expression or activation leve is at least abo 0,2. 2 , 3, 4, 45, 5,566.5, 75, 99 10 15 20, 25 30, 35,40, 45, 50,or 100-id ower(e.g. about L5-3 2-3, 2-4, 2-5. 240, 2-20,250, 34,340, -20, 3'-50, 4~, 4~10. 4-20,:. 4-5), 5-0 N 5, -2U, or 5-50-fold lower) ttan the reference expression or activation level of the co rrponding BER2 signaling pathway component in a z5 compound-senstive e (e, BT-474 4) treated with the compod, in a compound resistant cell (emg, lT/RS cl) tr-eaed with the compomd, or in a cell (e2. a tninor cell such as a breast cancer cell, itaa ticket cell, or a H-ER2-express'mng tumor ell obtained' from a patient. samnple) not treated it h the cornpound, ii11 in somse embcdments, the presence of a higher level of HER2 or p951 ER2 30 activaton in the cellular extract compad to the reference activation level oflER2 or p)95.HER2 in a compWound-sensitIvei cell indicates that the tumor (a .g, breast tumor or gastric tumor) is not likely to respond to the compoud (e., the turnor has a decreased likelihood of .respose to the compound i other emboiments, dh pes ft a similar or )ower level of BER12 or p9511ER2 activation in. the cellular extract compared to the reference activation level of HER2~ or p95hEFR2 in a comrpound-setsitive cell indicates that the tumor (et breast tumor or gastric tuor) is likely oto o the compound (eg, the tumx has an iWcreased likelihood ofresponse to the compound), h One Cmbodisnenft the level of HER512 activation in the cellulr extract is at least 2 to 3ASld higher than the reference activation level of ER2 in a comnpound-sensitive cell (e g, iT-474 cell), in another emibodimient, the level of p95'ER2 activation in the cellular extract is at least 5-fold i than the reference acivation level of p9ER2 in a c mpound-siti we cel (eg BT-474 cell), 10 j01624 In some embodiments, the presence of a lower level of HER2 or p9$HER2 activation in the celldar extract compared to the referencee activationt level atf H ER2 or p9$HER2 in a compound-resistant cell indicates that the tunr (e~g, breast tumor or gastric tumor) is likelyto respond to the compound (eg, the tumor has an increased likelihood of response to the compound), Int other enbodimnents. the presence of a similar or higher level 1$ of HEIIR2 or p95IER2 activation in the celluda extact compared to the reference activation level oflifHER2 or p9$THER2 in a compound-mcsistant cell indicates that the tumor (e~g breast Lunor or gastei tumor) is not likely to respond to the compound (e. the tumor has a decreased likelihood of response to the compound). 101631 in son embodients, the presence of a iower level of HER2 or p95ER2 20 activation in the cellular extract compared to the reference activation level of HER2 or p95HER2 in a cell (eg a tumor cell such as a breast. cancer cl, a gatric cancer cell, ox a HE2expressing tumor cell obtained from a patient sample)not treated with the compound indicates that the tumor (3 g. breast tumor or gastric tumor) is likely to respond to the comnpourid (e~g the mumor has an increased likelihood of response to the comnpmmd), Tn 25 other embodiments, the presence of a similar or higher level of HE1R2 or p95HER2 activation in the celular extract compared to the reference activation leve of HER2 or p95HE2 in. a cell not treated with the compound indicates that the tumor (eg breast tumor or gastric umor) is not likey to respond to the compound (eg, the tumor has a decreased likelihood of response to the compound). 40 I91647 i certain ensibeimn ts, the method comprises detemranring thecatiution level of both HE2and pP5HERUin the ecunr etrac In paticnar embodent te activate level of HE2R2 or opS lR comprises phosphorylation level of H o p93HER2 43 [1'65J In cerain other embhodiments, the method further cotuprises determirnn the activation level of one or more additional sigsal transductio molecues in the cellular extract Non-imiting examples of additional signal transduction molecules include EGIPR (HRlt FERt hE R4, P.SK, AKT IT, METNSOK3 4EMiPL ERK2 (MAPKIX), RK l l(MAPK3), PDKL P7i06K, GSK-3 , SHC, . R c4MET, cIT VEGFRL VE2 VEGR3, receptor dimers (ag, p95iER2/ER3 heterodimer. HER2/HER2 homoies PER2/IIR3 hetomner, HERI J/R2 heterodiner and/or HER2{HER3 heterodimer), and comintons thereof In patiar emodiments, the activation levels of one or more of the additional signal ttaNduction molecules comprise phosphorylation levels of uch molecules, 10 n father embodimens the method comprises determining the activation level of 1hR2 and/or p95IER2 in cmination wgith one or more oflHER3. P13K, and!oN p9HER2/HER3 terodimer in the cellular extra, 6 i some embodiments, the method further or alternatively comprises determioing the acivation level of one or more of H ER3, PDK, and/or p05HER2/IER3 heterodimer, li 5 certain inances Ahe p ceof a highr leve, of HEr, P13K. or p95hoER2/HER heterodimer activation in the cellular tract compared to the reference activation levdl of HER PD3Kor p R2/iER3 .heterodimmer in a compound-senitivse cele indicates thad the mor (e.gz* breast tumor or gastric tumor) is niot Jikely to respond to the cOmpoumnd (ag, the tumor has a decreased likelihood of msponse tor the coimpound), lan other instance, the 2W presence of a similar or lower level of' HERl), P13K. or rtp5HE2/HEClR3 beterodimer activation in the cellular extract compared to the reference ativtion level of HER3, PDK, or p95HEiR2/HER3 heterodimoer in a compiound.-sensitivc cell indicats that the tumor (e~g., breast tumor or gasric ummor) is likely to respond to the compound (ag,. the rtur)a an increased likelihood of response to the romond). In one embodiment, the level of H ER3 25 activation ha the cellar extract is at leat 2 to 3-fol higher than the reference activation level of HER3 in a comspouwdsensiie cel (eg, BT474 cell WI1671 in cerNn instances, the presence of a lower level of ER3, PK., or p95HIER2/HER3 heterodimver activation in the cellular extract wmpnaredl to the referce activation level of HIER3. P13K, or rp95RIER2/HER3 heterodimerin a camtpon-resistant cell 30) idicates that the tumor tag., breast tumor or gastric tumor) is likely to respond to the comph~ound (e g, dhe tumor Ias an increased likelihood of response to the compound), in other instances, the presence of a similar or higher level of HiER3, P13K. or p95H:ER2/IIEI heterodimerzactivation in the cehrd at extract compared to the referenc a'tivation level of 44 1CR3 91.2o 95-lnK?/lER3 heterodinmer in a comoutnd-re karantelld icates that fhe to ( ast tumor or gastric tumor) in likely toespond to the compound eg te tumor har adeceamed thehbood of response to the compond) P1164S In Sme instances, the presence of a lower level of HER3, P13K or 5 p95HtER2/HER3 heterodimer activation in the cehlr eract comared to thi efbe activation leve2 ofMHER3 P13K, or p95UER2/HR3 heterodiimer in a cex (eIg I tumnor cell such as a. breast canceceUt a gastrc cancer cellt or a lERl 2-expressing t umoar cell obtained. from a patient sampl) not treated wah the compound indicates that the nmor ( , breast tumor or gastrie rumor) is likely to respond to the compound (eg, the tumor has an increased ID likelihood of response to the comtpound), in other instances, the presence of a similar or higher level of.HER3, P13K, or p9$HER2/HER3 heterodimer activation in the duar extract compared to the reference actlyationt level of HEI~R3, P13K, or p95.RER2/HER3i hetrodlimer in a cell not treated with the. comopotund indicates that the tumor (je. g, breast tumor or gastric tumor) is not likely to respond to the compound (eg, the tumor has a decreased likelihood of' n, response to the compotd) (0169 %in some embodiments, the cell (ae~s, the test cell fromn which a celladar extract is produced) is a tumor cell such as a breast cancer cell, a gastric cancer cell, and/or aHE2 expressing tumor cell in certain instances, the tumor cell is a circulating tumor cell or a fine needle aspirate (F 2 NA) cell obtained &rom a tumor, In other emubodiments, the cell (a g, thre 2t rest cell frn which a cellular extract is produced) is isolated from a sample that is obtained, apg, from a. breast or gastric cancer patient NondimiJting examples of samples include bodily fhlid samrples such as, for example, a whole blood, serum, plasma. ductal lavaige f'luid, nipple asp iate.,1 iyph, bone marrow aspiae, urane, saliva, and/or tide needle aspirate (FNA) sample, in oarticular embodiments, the sample comprises a whole blood. serum, plasma. 25 and/or turmor tissue sample such as breast er gastric moor tissue or HiERZ2-expressing tumor {0170Q in cerrti instances, the method may further comprise the step (e) of providing the resu aof the comparison obtained in step (d) to a user (aeg, a clineian such as an oncologist or a genei practitioner in a. readable ramat, in some instances,. the method may fUrther 31 comp6ise sending or reporting the resut of the cmparison obtained in step (d) to clinician ne:, an oncologist or a general practitioner, ln other instances, the method mau further 45 coropde rewcrdng or storing the result of'the comparison obtained in step (d) in a computer database or other suiltbe machine or device for storng informatiot eg., at a larory. [17) in. some embodimnts, dotemdining the activation evel of one or more ER2 siaing pathway omponems (e ilER2, p95HiER2 IHR3.PIS1 p9ERZ ER2/UiER HER 4, and/or SHC) in Stp (C composes detectin a phosphoryhmion level of one or more HER2 signing pathway components in the elhuar etet with antodies pci c tor the phosphorylated fcrm of each of te 1{ER2 signaling pathway zonponents to be detected 101721 Activaton (eg, phosphboiatkin) levels and/or status can be determined using any of a variety of techniques. In paticular m ients, the activation (e<g> phosphorytion) c0 level and/or stams of one or more }12R2 signalig mtway componets (e g, HER2, p95HE9R2, HER3, P13 , p95HER2/HE/R3 .Ih ER1, and/ SH8IC) in step (c) is detected with an immnunoassay such as a single detection assay or a~ proxihy dual detection assay (g a GQO1aborative Froximtity I munorissay (COPtA)) as described herein. (01 73j In yet another aspeet, the present invention provides a method for mnonitoriang the ii resonse to therapy with a compound that modulates HER2 activity in a subject having a tumor and. receiving therapy With the compound. the method comprisi~ng: (a) lysing a cel obtained itom the tumor to produce a cellidar exterct: (b) determining the expression and/or activon (e g,. phosphorytationf) level of' one or more components of a HER2Z signacling pathway in the cellular 2i extract; and (c) comparing the expression and/or activation level of the one or more components of the HER2S signaling. pathway deteeanined in step (b) to a reference expression and/or activation level of the one or more components of the HEZPc2 signalin pathwvav 25 wherein a diffneee between. dhe expression and/or acti vmran level of the one oramore comaponents of the HER2C cignaimng pathway dietermined in. step (b>) and the referene expressiont and/or activation kevel of the one or more conmponents of /te HER2 signaling pathway indicates that therapy with the cotmpotund should he continued or adjusted (Qg maintainig the current dose of the compound, changing a subsequent dose of the compound 30or selecting arn alternative anticancer draug 46 j74j \rOprerre asp'ecsethe nremn invention provides a nmethod for nTOZonIICn th responv to therapywtha comnpundibat t dAates .11R2 activii da subject havig a umor and re en..trapy whb the nmposd the method eomprhin (a) lysing a ;dR obtainedIrtte tumor to produce a cellular extract 5) dteamining the activation level f HER2o ep itn the ceular extract arnd comparingng the activation level of HER2 or p95FER2 determined in sep (b) to a. relecuce activation level of HER2 or p9511EiRz wherein the presence of' a higher level of HEIR2 or p95HlER2 activation in the 10 celhtlar extract compared to the refeence activation level of fHER2 or p95HER2 indicates that thempy with rhe compound shoul be adjusted (e.g. changing a subsequem dose of the compound or selecting an aternative anticanicer drug). t1017$1 Non-limaing examplesC ox cormpouinds that modulate HER2 activity include monoelonal artbodies, tyrosine knase inhibitors and combinations thereoft in prefrered i ehmbodinent\ the HER2-modulating compound inbit HER2 activity dlor bocks HER'2 signaling, e s a HER2 inhibitor, Examples of HER2 inhibitors incald, but am not limited t, maonocional anntbodies such as nstunoab (Herceptint) and pertumab (2C4); small molecule tyrosine kinae inhibitors such asgfitinih (Iresai edotinita (Tareeva) iltnb CP-654577, CP-72.474, canertinia (CI I33), HKI-272, lapatinib (GW-$720 16; Tyket") 20 PK 6, AE788, S 9966 KI37, BIBW 2992ARRY-3454J 643 and lJb26483327;m ad. combinations thereof In other embodinmcta, the HER2-m~coalting compound activates the HER2 pathway, eg., i a iIER2 raca [01761 i some embodimenta, the reference expression or activation level ofone or more componens of the HER2 signaling pahway (4>. H2R p95HR2\ HER3, PDK. 25 p95HER2/HER3, HERI, SBC, etc) is obtained &on a cell sensitive to the compound (e~g, a .HerceptirVsensitive cell) that treated with the compound (e.g. Hereeptiri). iLa certain emtbiments the ell entiv to the compound (to, compound.sensitive cell) is seleeid from the group consisting of aBT474 cell, SKBR3 cell, M27 celt MDAMB-36Tt celh UACC412 cell, UACC-93 cell MDA;MBEV?7 celL SUMt 9 celi. SUM225 cell, N87 cell 30 OE19 cell and combinations thereot in some instances, the compound-sensitie ceB is engineered fAn, an existing cell or cell line (eg, a coUpound-aesistant cell or cell linj to create a cell r cell line that is sensitive to the compound (eg, by expressing a HER2 sigaling pathway component (eg, HER2) modulated. by the compound in the cell or cell 47 ine Pre 7 b7. Ay, h m asitive cell is a oer cpt n en shive een. suII as a 1T474 (tU77 in. other embodimnents, the reference expression or activation levei of oneS Or more components ofll IthIR2signalng pathway (eig., HEiR2, p91iER2. 11ER3, PJGK, Sp95ER 21ER3, HER, SEC, en) is obtained from a cell resistant to the copound (e.g a iHerceptuin*-resistant cell that is treated with the compound (e~vg, Rercrepti*. In certain m ,e cell that. is rsistant to the compoud (ie compoumdresistant cell) is selected from the group consisting of a 13T/R cell, .MDAMB-23I1 cell, SKBR13/IGF-AR (Al:, IMT-1I cell, BT474/HR20 ceL SKBR3/P2 cell, NH129 Cl. N.H47 cel MCI>? cei MCF 10 7/713 cell, MCF7/HER2AW cell ZR&5-I cei. BT20 cell MDAd-MB-435 ccli T47D cell MDlA-MB-43I3 cell. MDA-MB3468 cell. CAMA1 celL. MDA-MB-157 eell, EMI92A cell Rll cell EM19 cell, CALSI cell, NTC3t cell, N C4 cellS PU97 cell S 16 cll and cobmatons there hio In so ime stands, the compound-esit ell is engineered from an existing celI or cell line (eat, a compoundeitive cell or cell line) to create a cald or cell 1 n lne that is resistant to the compound (e. by knocking out a HER2 signaling pathway component (g, NER2) modulated by the compound in the cell or cell line), Preferabiy, the compound-resistant ceil is a iierceptin*resistant cel such as a BT/R celL [0178m ) n trther embodiments, the reference expression or activation level oF one or more compons ofrthe HER2 signng pathway neg. HER2, p9oHER2, HER3, Pin 20 p951ER2/HER3 PER1., S1HC dc,) is obtained from a cfl (.g{, a tumor ceil such as a breast cancer celil a gastric cancer cell, or a lHER2-expressing tmoor cell obtained front a patient sample) that is not treated with the compound (. g, Hereptirf). hi particulAr ntodiments, he cell that is ntot treated with the coanpound is obtained rom the same sample that the isolated cell (ags, a test cell to be i nterrogated) used to produce the eljlular extrt is 2O obtainuedk t1 79) in certain embodiment s, a higher level of' expression or activation of a HER2Z signaling pathway comupon entl (exg HE R2k, p95 R2, 11R3 P31 p5HRHE3 HERL SHC, etc) is considered to be present in a cellular extract when the expression or activation level is at least about L5, 2,2.5,T 15,4 4.5,5, 3 6, , 7.5, 8, &5, 9, 9,5, 0 10 1 W, 2(h 2, 30, 35440, 45, 50, or 100~fol higher (e. abot 1,, 4,2-5,2-,2 20, 2-50, 3-5 3-410, 3-:20, 3-50, 4-5, 4-10, 4-20, 41.0. 5-10, 5,4$, 5-20, or 5$~50-foid higher) than the reference exresion or activation level of the corresponding HER2li signaling 418 parbwvay ccmpanent n ar con mpoandscsitive cell Tg Q 474 ce) treated vh the comTpound~ in a comipoundgresistant ell (« B 1/1 cell) tread wiah tecamnpoundtor ina cell (g. atmmor cell suca breatcancer l.a aade cancced or HE2apesaing tainor cell obtained Mm a patdera tample nottreated vith the compound. 5 [US k",*-- of*W ofv a~Ot~ HEyt~toa R2 siaalin I OIl*1 In other embodiments a 1ower level ofeeso oratvtono E2 inln pathway component (g,- HER2 p951ER2, HER3 P13, p95HER2/H ERt HERi, SHC, etc) is considered to be present in a celuli et when the expression or activation level is least about 1, 2,2,5, 3,15, 4 .5, A 5 6 5, ? 5 9 9.5, 10 15 20. 25, 30, 35, 40, 45. .50, or 00-fold lower (a. g about LSA3 2~3, 2-4. 2-5, 20, 2-2T 25, A-5, 3-10, 10 3-20 -5, -, 4 10, 4-20, 45 5-10, G -15, 5~20, or -50-foid lower) than the reference capssio or acivion level cfte corresponding 0HERt2 signaling pathway componem in a cormud-sensv cell (egz, BT-474 cell) treated with the comnpound, in a compound resistat cell (eg- BT/R cell) tested with the ompoumd, or in a cil (eg, a tumor el such as a breast cancer cel, a gamc cancer uci, or a HER2 xpressig tumor cell obtained from a 5 patient sam) not treated with the comp9ounld. [11M in. some embodiments, the presence of a higher level of HER12 or p9513ER12 activation in the cellular extract compared to the reference activation level of HER2 or p95HER2 in a compoun-sentainve cell indicates that therapy with the compound should be adjusted (e g. changing a subsequent dose of the compound by increasing or decreasing the 20 subsequent dose or <lectnng an alternative aniac~ ) n oter embhodimnts, te presence or a similar or lower level of HER32 or p95HER2 activation in the cellular extract compared to the reference activation level of HR2 or p95HER 2 in a componsenstive cell indicates that therapy with the enmipound shcrald be continued (eag, maintaining the currem dose of the compound). In one embodiment, the evel of HER2 activatio in the 25 celhdar extract is at least 2 to 3-fid higher than the reference actvation level of HER2 in a conmpoundSensitdve cell test BT-474 ceil)< In another embodiment the level of p95HERi2 activation in the celluoaro e:tract js at least 5~foid higher than the reference activation level of p95HER12 in a conmpound-sensitive cell Leg> BT9-474 cell). pn82j i acuteie entodnents, he isesencee of adlower level f 11ER2 or p95HER 1 2 30 actiation in the cel sha extract compared to the reference activation level of I19-R2 Or p951HER2 ina eompound resitant cell ial aes thattherapy with the compound should be continued (aeg maintaining the carrnt dos of the carnpotmd). ii oher enmbodinits the 49 presence of a similar or higher level of HER{2 or p95HEtR2 activation in the cellar extract compared to thtsfrc acdivation level of I-ER2 or p9514ER2 in a comnpoundtSeStanht cell Julth 83ifcreth niates. tha therapy with the compound should be adjusted (eg, hanging a subsequent dose ofthe copoundi by mtereasing or decreasing the subsequent dose or selecting a 0 dteroative anticancer drug,. [13) in some embodimens, the pre sence of a lower level of BLER2 or p95.iER2 atct vation in the ceihaiar extract cornpared tos the reference activation level of HER12 or p95H ER2 in a cell (e&g, a nunor cell such as a breast cancer cll, a gastric cancer celt or a HEtR2txpressing tuor cell obtained froma a patient sample) not treated with the compound 10 indicates that therapy with the compound should be continued (e. g, aimaining the current dose ofIthe tompound), in other embodimeas, the presence of a smlr hg level of HR2or p95H1ER2 vacation itn the ceilhdar extract compared to the reference activation level of R2 or p95HER2 in a cell not treated with the compound indicates that therapy with the compound should be adjted (esg, changing a subseqneunt dose of the comrpoad by increasing or decreasing the subsequent dose or selecting an alternative anticancer drg). 101341 in certain. embodients, the method comprises determining the actioaion level of both HER? and 995tlER2 in the cellular extract In paticlar embodiments4he activation level of HER2 or p95HERt2 nomprises a phosphorylation level of HER2 or p9HER2. 1S8J in certain outhe embodiments, the tuethod Ifuder comprises determining the 2 activation level of one or more additional signal transduction molecules in the cellular extrt~~ Nod d0 ogP" examples OF additOmd signal trasduct ion molecules include EGFR (HFRTO F HER3, HR4, P3K, AKT M& K, PTEN, StK3, 4-BVP R EK2(MAtPK4 ERKI (MASPK3)1 PDKIL P70S6K, GSIv3f. SHC, OGEUt, c-MET, c-KIT. VEGFRL VEG-R2, VBGR3 reC'~ ceptor dimsrr' (evg 95%iER2A/BER3 heterodimer, t(ER24HER2 hamdimer, 23 14ER2/HER 3 heterodhmer ItiI HE RHR2 heterodinmer, and/or H4ER2/HESR3 heterodimerr), and combimations thereof. in particular embodiment, the activation lvels ot one or mare of the additioal signal transduction molecules comprise phosphory lationm levels of suich molecules, In further embodiments, he method. comprises detenmning the activation level of HER12 and/or p95HR2Z in combination with one or more of HERI~ 3, <K and/or p95HliER2/HtER3 31 hetemdtimer in the cellular extinct. 86 nsem nbodinmenls themehod thiher or alternatively comprus adetermining the activattion level of one ormom of HlER3 P3K, ad/or p9)lHR2/IIERSheterodimer. i mAUCRS ~un50s certain nstanes, the presence of a higher level of iFR3, P31K, or p95HER2/HER3 heterodimer activation in the cellular extract compared to the reference activation level of ER<3, P13K. or p9HSE MR21hER3 heterodir in a compomd-sensitive cel indicates that they wit the compound should he adjustd et g, changing a subsequent dose of the S compound by increasing or dereain the subsequent dose or selecting an ahemaative anticancer drug), in other instances, the psee of a similar or lower level of HER3, P13K, or p95HIER2/HER3 heteroduner activation in hee cellular extrct compared to the referee activation level of HER3 PUT, or p95HER2/HER3 heterodmer in mp ds Ntive ce indicates that therapy wit the compound should he contnued (eg maintaimig the 10 current dose of the cnopamrd), In one ebdmnthe level of 1HER3 activation in the ellular extmot is at least 2 to 3old higher than the reference activation level of HER3 in a comtpound sensitive cell (eg ls ~474 cell). 0187} In certam instantes the presence of a lower level of ER3, PDK, or pPtfER2/lER3 heie dimor activation in the cellular extract compared to the reference $ activation leve Wf 1ER3, P13K, or p95RER2/1EP3 heterodimer in acompowd-resistant cell indicates that thcrpy vith thne compotmd abould be continued (g mainanna the current dose of te cmunumd), bI other instances, the Ipresence of a similar or higher lcycl of HERI3, P13K, or p95RER2/HERi3 heterodhimer activation in the cellular extract compared to the reference activation level of HER3. P13K, or p95HfER2/i-E'R3 heeoie i n a compound 20 resistant cell indicates that therapy with the compound should be adjusted (cg changing a subsquent dose of the compound by increasing or decreasing the subsequent dose or selecting atn alternative anticancer drug)t [0188 In sone instances. the presence of a lower level of.HER * P13K, or p95ER2/.IR3 hetemdi nNr activation in the cellular extract compare to the reference 25 activation level oftHTER3, P13K, or p95HER2/H1ER3 heterodimner in a cell (e4g., a tumor cell sh as breast cancer cel, a gastric ancer ce or aHERaed tromt a patient sample) not treated with the comupounad indicattes thatt therapy with the compotud should be continued (e.g. maintaining the current dose of the compound). In other instances, the presence of a similar or higher level of IiER3<, PD3K, or p95U.ER2/HLER3 30 heterodimer activation in fhe cebr ext compared to the rcfrence activation level of HER3, P13K, or p95HER2/HER3 hteer i'n Wacel not treated with the compound indicates that therapy with the compound should be adjusted ( g, ranging a susqun 51 dosea othe contpound by natsing or decreasing the sabsequrent dose~ or selecting anI alterntive anticancer dru gt [1I89] hi some embodnems, the cel (eg the test cell from which a cellar extract is produced) is a tuor cell such as a breast cancer cell a gastric cancer cell or a HER2 expressing tmor ce, in certn instances the tinor cell is a circulang rumor cell or a fine needle aspimte (FNA) cell obtained for a umor. in other embodiments, the cll (e-. the test cell from which a cellular extract is produced) is isolated rn a sample that is obtained, eag, from a breast' atric cancer patient Nonimiting examples ofsamples include bodily fluid Samples such as, for example, a 'hole blood, senm" plasma, ductal lavage fluid nipple ) aspirate 4 lymph, bone marw aspirate, uine, saliva, and/or fine needle aspirate (FNA) sample. in particuar embodinents. the sample comprises a whole blood, serum. plasma, and/or tumor tissue sample such as breast or gCatrN tumor tissue or HER2-expressig tumor tisse. [019) ia certain instances the method may tiarther comprise the step (d) of providing the I5 result of the comparison obtained in step (c) to a user (eg. a clinician such as an oncologi or a genera practitioner) in a readable ora In some instances. the method may further emprie Sending or reporting the result of the comparison obtained in step (c) to a clincian, e.g, an oncologist or a general practitioner. in otter instances, the method may frher compirise recording or storing the result of the comparison obtained in step ()int a computer 20 database or other suiable machine or device for storinI information, a g, at a lahoAtory. If9f} in some enmbodiments) determining the activation leve of'one or more IER2 raig pathway components (e.g, HER2R p95HER2, ER3, P13K p95HR{2/'lER13, ERl and/or P SHC) in step (h) comprises detecting a phosphoryluinn level of one or more HER2 siAali ptvhway cowponems hi the cellular extract with antibodies specific :kr the 25 phosphorylated ton of each of the HER2 signaling pathway components to be detected [1921 AcIvaion (. g, phosphotylation) levels and/or status can be determined using any of a variety of techniques in particular embodiments, the activation (ipg, photsy mat) level and/or status of one or ore HER2 signaling pathway components (eg, IER2, p95HER2, HER3, P3K, p95 HE I2/HE3, HEl, and/or SHC) in step (b)is detected wit an 30 immunoas'say such as a single detection assay or a proximity dual detection assay (e. a Cflliaborative .Proxinmity Inamunotssay (COPtIA)) as described herein.

jai931 In a luher aspect, the present invtion provides a method for onitoring the HER2 stains of a subjct with an iratiai }ER2-neative primary breast tumor, the method compnrisng: determining die HER2 sttus of circulating cells of a. solid tumor obtained 5 rom the subject by detecting the presence of activated HER2 in the circulating cells, wherein the presence of activated HER2 in the circulating cels indicates a conversion fromt a HER% negative status of the subject to aiHER2~positive status, [01941 In one mbeubject (g, bnum) has metastani breast cancer, in anothler embodiment, the circulating cels ofa solid tumor are selected from the group it consisting of circulating tumor cells, cruag endothelial cells, circulating endothelial pigenitor cells, cancer stem cells, disseminated tuor cells. and combinations thereof In a futher embodimenh the ER2 stams of the primary breast tumor is determined prior to deternining the kHR2 s f creating cells. {01901 In certain embodimens, the presence of activated HER2 in the circuatbag. cells is 15 asciated with responsiveness of the subject to treatment with a compound that modulates HER2 activity, Nonlimiting eCampes of compounds that modulate HER2 activity include mnenacional antibodies. tyrosine inase inhibitors, and combinations thereof In prefrred embodimoents, the HER2-mrodulating compound inhibits HER 'ctivity' and/or blocks HEPR2 signaling, eg, is a HER2 inhibitor. Examples of iER2 inhibitors include, but are not limited 20 to, monoelonal antibodies such as tasuazumab (Hexceptin*) and pertumab (2C4)small molecule tyrosine kinase inibtors such as gefitinib (ressa", erloti (Tarceva') plihinib. CP-65577 CP-24714, canertinib (CI 1 033), HKP-272, lapatinib (G\W572016 ; Tykerf), PKI.-I66, AEEZ788, BM8-59626, HK-3 57, BBW 2992, ARRY-334543 JNJ-26483327, and. TJJ26483327; an~d combinations thereoc toI other embhodimnents, the iHR2-miodudaing 25 compound activates the liftR2 pathway, e.g. is a HER2 activator, 10196j in some embodiments, the methods of the present invention may fther comprise the step of obtaining a sample from a subject having a breast tumor from which cells of a breast turor are isolated, The sample may be obtained from a breast cancer subject before treatment with a .HER2modulating compound and/or after administration of a HER2 30 modulating compound (e g: at any time throughout the course of cancer treatmem). Suitable sampes inclde, but arc not limited to, whole blood, senum, plasma, and cormbnations thereof In one preferred embodiment, the sample is a whole blood sample, in this enbodiment, ciradating cells of at breast tutnor rnay be isolated. from the whole blood sample, if isolated cell are obtained frm a subject who has niot received treatment oit a HER2-moduating compound, dhe isolated 2clrs may be incubated in vrito under suitable conditions with one or a cocktail of HER2moduading coronds. 3 [16971 Circulating cells of a breast tunor may be isolated fr a sample by any techique known in the at efg, by imnmomnagnetic separation, th.e Cel Tracks 5 Sytm icolii separation. PACS, density gradiemt centrifhgation, and depletion methods. isolated cells such as cirlatng cells may be lysed to thereby transform the isolated cells into a cealniar extract by any technique known in the art, i 10198j in other ebodimentc de method further comprises detcmintng the status (eg, activation level or state) of one or morddiional signal transduction molecules in circulatNig cells of a solxd nior (eg" in a cellular e(raCt produced fro ling the circulating ce) Nonlimiting examples of additonal signal tansduction molecules snchude components of the H{ER2 sigrnling pathw~ay such as, ea. p9HER2, ER (RERD) 5 HER3 1 HER? PDK, AKT, MElK, PTEN, SGKS. 4&EP l, ERK2 (M K I) ERKI (MAPK3s PDK1, P7056K, OK-, RS1.C, IGFIR, 11ME ef-i V VG R i, VEG'R2, VEGFR receptor dimers (e.g, p95HER2/flER3 heicrdimer, HER2/RE R2 homodimer, HER2 RZ-3 heterodimPer, HER i /HER2 heterodimer, and/or H1 ER2/HER3 betrodiwer), and combinations thereof in paricular emboiments, the activation levels of HER2 andor one or more of the 20 additional signal transduction molecules comprise ph.osphoyiation lares of such molecules, f{l 99J Activation (e~g. phosphorylationr) icevels and/or status cia. be determined using any of a variety of techniques, in some emrbodinmens he activation (e.g. phosphorvtatian) level andior status ofYHER2 in circulating eeJs of a solid tumor is detected with an immunoasay such as a single detection assay or a proinity dual detection assay (eg a COl aborative 25 Proximity lmmunoAssay (COPA)) as described heein, 102U01 In partcular embodi mets, the presence of activated I3ER2 is detected using an mmun)assay combprsing: (i) incubating a celar extract of the circulating coils with a dilution series of capture antibodies specific for HEiR2 to fann a plurality of captured antalytes, 30 wherein the capture antibodies ate restrained on a solid support; (ii) lucubating the plurality of captured arnalytes with detection antibodies conmprising activation stateiadependent: antibodies andt activation state~ 54 dseedcotlhodien petdie folbsR toto a pluralit f detectable captured analyses, vherein the activation stateiependent antibodies are labeled with a facilitatings 1io iety, the activation staiedependen antibodIes are labeled with a first -ailtalnit~ta member of a signal amplicton pair and the ailt ey gee rats an oxidizing agent which channels tn and reacts with the s m0embgr of the signal amplification pai; (ini) incUbating the plurality of detectabk captured anaytes with a second member oothe signal ampticaton pair to generated amplified signal; and iv) detecting the amplified signal generated from the first and second members of the signal amplification pair. In21]l cutain instances, the method my further comprise the step of providing the result ofthe 1fER2 status determxination to a user (e,gz a. clinician such as an oncologist or a gener al practitioner) in a readable .ibrmnat. in some ins tances, dt method may farther comnprse sending or reporting the result of the HER2. status determination oi a clinical eg man oncologist or a general prantioner, In other instances, the method may further comprise recording or storing the result ofthe HER2 status determination in computer database or o'ter suitable machine or device for storing info rmation, e gm at. a iaboratory. W2621 hn anadditional aspect sh preset itvemion provides a me thod for selecting a 20 satable anticancer drg tb'te treatment of a breasittumor, the wetlhodcomprising () contacting a ellbtainedkfom a fne needle aspirate (EN) samspk o he manortwith an anticancerdrug; (ba) lysing the cell to produce a cellular extract; (t) deterring the expression anc/or acivation level of one or more signal 25 transduction molecules in the cellular extat: and (d) comaparingy the expression and/or activation level of the one or mote signal tiansducticm molecules determined in step (e) to a ref erenace expressiont and/or activation level of the one or more signail transduction molecules, wherein a. diffluence between the expression and/o~r activation level &fdte one 30 or more siganal transduction molecules determined, in step (e) andt the reference expression and/or activation. level of the one or more signal transduction molecules indicates that the anticancer drug is suitable or unsutable tbr the treatment of the breast tumor, [ i a paculdar emoict the meod for selecting a suitable anticancer drug for the treatment. of a breast tuor compries: (a) cotacinIg a. cell obtained from a fin needle aspirate (FNA) sampe of the tumor with an anticancer drug; (b) lying the cell to produce a cellular extract; (g.) determintig the activation level of one or more signal traasduction ol in the cellar extract: and (d) comparing the activation level of the one or more sigal transducton molecues dctenmined in. step (c) to a rference activation level of the one 10 or more signal transduction m !oecu-es generated in the absene of the anticancer drng whereirthe presenceofa bstandaly deceased aathi on kei of one or more? nf the slgnal transductionioleculs itt We cellhar xitrasct compared to the reference act vationevel of the one or more signal transduiction molecues indicates that the anticancer 5S drug is suitable for the fretnento the breast tanor. 02Z*4$ in. samec enmbodiments. the aied vation level of a signal transdudimon moeculde is considered to be '"substantiaIny deczrea sed" in the presence of an anticancer drug when it it least about 50% , 60%, 65%, 70%, 75%, 80% 85% 90%, or 95% less activated than in the absence of she antwicer drug, in other emnbodimntsuN the ai.dv.ation level of a signal 20 transduction molecule i s considered to be Nbstantially deerezald inthe ptsence of an anticancer dhug (I) when there is a change fr m high or strong 'atIvation of the signal tansducion molcule without the antiancer drug to medium, weak tow or vcry weak activation of the signal transduction molecule with the anticancer drug, or (2 when there is a change from medium activation of to wransducion molecule wihut the amcancer 25 drugstoweak, low, or very weak ativIon of a the signal transdtiton moecule with the anticancer duin )2O5] in one emabodinanit the .F. sample is btained font a.subjecste~. hmturt wth meta static beast cancer, hnumother embtodinente methodi further comprisceshe step of adminiteriag the anicancer drug when.the anticancer drug is idenmifed as being suitable for 30 the traant of the breast tumor 111656 ee ~(I%6 in ne etnbdnea. the tieancer ugonprssan ~gettt that itntrferes with the Ict on activated signal trdndpat Wa onerns in. encer oS Non inooing exanpa d uch agents Pinlude those tededow in Table EGFR (ErbB1) (A) HER4 (UE) (C) HER-3 (ErbB3) (E HER (EMtS4 taroet C uximabTrtuuaAnsdU) Paniturnumab (HerepttA") Matuzumab Pertuua 24 *Imeterodimerzatio NER 2 Phs6i' EGFR (Erb1 (ER4 2} (D}, ErbBIt2 (F} ErbBi)24 JG) EraPnhC-724714 (Pfizer) Lapatinib (TykerV) Cartnb G2eftinib HIKI-272* ARRY-334543 EKB R * HAK7 (PreciIcal) JNJ-26483327 CL-3.7.86" --- l-W 2_12 JNJ-2l -47 WVyethl Preversible, 1/iI (Wyevc~h rreversible. i NSCLC, treast CRC * soehringer *Pflzer, !trsversible, 11 ",(Wyeth, ire vrs bngelhe, Irreversible, NCLC, Breast Preclinical) VII Prostate. Ovarian __ Breast Rat (H) SRC (H)l Mek: (I) W PkB~kB (i) Soratberh AZ P'fS2590' (f NSCLC) PLXA. 32 (Plex..kon) j.AZD.244 -- A- -a-Az XL518 Exetsis1DNA _________ rPK VE(JFR and VEGFRl: IRa& 051 :Everochmusx PX-866 4 ' 'Aetin (DA AC)27 (KCC Temsrlimus" HM 3 MG-7O6 (t 90GER) AP,25373\* VFGPTrap* t verobnms (Novarts, com thnation with Gefetinib/Erlotlnib. Wi: NSCLC, GlNoblastoma) (PDL) antVEGFa *"Temstrohmimis (WytVeeernAeh combinationr wlth NrlxPsCC a rthi (Receto miic Gefelinib/Erlotinib'; /!l: (PnaLe 2) N$CLCZ G obatama) *P-2.t3 (Ari.4/. VEGFR2 target(L) EPH A-D DC1DP791 (UCB) IBa013P- 3y-579352 +PGFR) W(AKCMIR) IMC11I213 Fruy AG 7 3e** BMS-40215 (+R1\r~~ "huatoo! ~ s CD-9& -70lt (Esal) KRN-95I Pazopansb***OI-5**B1 03St930 (4 ct~it, ?DGER) 1mclone (Pmase 2/37) **himerc kgG1 taaist "OS PFiZER: f+ ErB + VEGFR2 PD{FR) (NSOLC, Ovarian PDCFRhe (NRCCha0 GSKMultpe **(VIEGFR1 .2 myetom, osvanRCC FGFR3.PDGFR) Phase 3 enrollment . ......... .............. _ ---- ---- __ _ __ _ _ VEGER 2Werseil2 VGF ({tFRTtE 12 cFMS, POGFRSM (M) ZD6474' STrafer(b*tFL TS) ACLG4'X Sunitinib GSK AZ (AZD2171) BM3 Novart (AEE-788 Am-gen ___Others I: thyrod6 NSCLC) EPMt-2). (Petient *(ROC, H CC, NSCOLC(tht resistant to E tiotinib: Melanoma(id)) As an patents) (Phase 2 NovatiG, PFaRe112 PDR, targt (P) AI targt (Q) FT 3 SE Nimctiib Datina NIlodnib AZD-053O it&;rgt (R) -GRFE1 AMG-70 Chiron Merck stzs XL9s_________________ Nova, I~~~~~~~ ~~~ --- ~- ------- - - - - --------- __ __ __ - - -- _ _ _ _ ---- __-----_ _------_ _-------------- -SP9r hbitor - Andt ict to Drugs: gOther taget ..5.4........erHDA inh-o- 17 AAG Pactxa t BCL2 \hnfastne< Viruistmie, Inmteaetc - ______________Proteoome~ nibitors~ ____ ,Micottbue stabilzer, Adjuyvarnt and advanced 4 Qfll4\~ breast caner: NSCLC, 'nI nay PH imAdognideedn mulemyelma- 4 (Microtuouule stabilzer; a posar U.) Adjuvant and ad ncead * Koan,1/ilsold 2Seast canaer, NSCLC QOatan cancer, AHDS related ________________ (M Z C ~rotuouLe Dea-stabdizers) _____ (307 n certa in emibodimecnts, the anticancter dmg comprises ea antti-sgaling agent (1 a, a cytostatie drug) such. as a mononlonal amtibody or a tyrosine kinase inhibitor; anan proliferative agent; a emnotherapeutic agent (La. a cytotoxie doug); a hormonal therapeutie S agent; a radiotherapeutc age; a vaccine; and/r any other compound with the ability k, reduce or abrogate the uncontoled growth of aberuwi. cel such as cancerous ells L ,ome embodimenis, the isolated cells are treated with one or more at-signaling agems, anti proiiferative agns and/or hormonal therapeutics agents in. combination with at least one chemothorapeutie agent 10 {O208 Examples of' anif-signasling agents suitable for use in the present invention include, without limvitaton, monoclonal antibodies such as trastuxumab (HeIceptn"), peruzanmL. (2C4). aicetuunabt Campath~~) bevacizumnab (A\vastin*)X ceuiab (BrbiltuO5 genAnab iMyswoarg t .panitun ab (VectibiC), rituximab (KR\an) and rositrnomab (BEX\AR*); tyrosie kioase inhibitors such as gefitinib Iressa"%, sunitinib 5 (S't -t*) ertinib (TarevaTh lapat inib (1W20t6; Tykerb' canertinib (Cl 1033) semaxinib (SU 16) vatiib (PTK?871ZK222584> sorfenb (BAY 43-69006; Nexavar" imati nib mesyat (Gle r evee ) letunide (Sil 1). vandetandh (2ACTYIAn; ZD6474), pilitinib, CP-6t4577 CKP-724714 H1K~272, PKI-166, ABE788, BMS-599626 , H 3KI57 BlBW 2992, ARRY~3'34543,f JN-2483327, and JNt2643327; and comnbinations themeofE 20 [02091 Exemplary antiproiferative- agents include mTOR inhibiors such as riaolirs frpayxcin), temsirolimus (CCi-779 and everolimus (RADOOD); AWT inhibitors such as iL6-hydroxymethyl-chirotinositol~2(RI-2-mtethy43--octadecy.-s-gyceocarbonate 9 59 metsoxy~ 2 mt hylelipticiniumn acetate, 1,3-ydro-1(1-((4-b-phenyH-i midazko (4 g'jquinoxa in-7-y1)ph-loylet>yn4 piperd inyI)-2H4benzi idazo-2-one, 10(4>'N~ diethylamnino~buty.)-2-chlorophenwxa:s:ine, 3-farmychromone thiose-miarbazone CuIU Qomplex), API2, a 15-mer peptide derived fom amnom aids 1024 of the prot-ncoene S TCLI (Hromnra at aL. Bioa Chem 279:534073418 (2004), KP37-1 and the comnpound described in Kcaikowski et a, J Am. Ch Sa, 12$T11441 .145 (2003') and at at. Cancer Ca, 4:63476 (2003); and comations thereof (0210o1 Nonimiting examples ofchm therap uti ants include piatimn-based drugs (atgg, cxaliplatin. cisplatin. carboplatin, spiroplatin, iproplatint satrapiadn, e.), aikylating agents (a g. cyclophosphamide, ifostmide, chlorambucil, busuWfartnmephalani mecrethamnine, uram~ustine, tiotepa, nitrso-nreas. ae),~ antimeabolites (eg. 5 fl:uorouracil, aaIhiopiSe, 6-mecrcaptopurine, me thotreate, Ieucoveria. capscitabine, eytarabie, lnxunidine, fludarabine, gemaitabin zar") pemetrexed (AIMTA raltitrexed, aet.), plant alkaloids (e.g, vincristine, vinblastne, vi webine, vindesine, 1$ podophylktoxin. paclitaxel (Tzaoit dcetaxel (Taxotere* etel topoisomase inIitrs (eg:t, Sotecan, topotecan, fmcrine etoposide (VP16), coposide phosphate, tenpcxide stae) antitumor antibiotics (e.g~: doxorubicin, adr imycin daunomi cia, epinibicin. aetnoroycin- bleoyin tmy cin, miteanttrda.e plicamycin, etc), phraceuticaliy acceptable salts thereof, stereoisomers thereo, derivatives thereof' analogs thereof, and 2.0 combinatons there [92 11] Exam~ples of hcornmna1lbheapeCti& aeni iluO~.vde ithut limritation, mals inhibitors (e.g, amnogutethiide, anastrozole (Arimidex"),c aeto oie (Femwr vamol exemSrfCtanel (A romasin%' 4-androstened6, 7-triosne (6~OXO%) 1,446- androstatrien 37 dione (A\TD), formestne (aL ntaronI ae), selective e atge receptor modulators (a g 25 bncmifene, fuett. lasefoxifene, ralaxiene, tamoxifen, toremyene etc ), nieds (mg, deasmethasone), finwsride, and gdrmrain hormone agonists (OnRI) such as goserelin, pharmaceuticaily acceptable sas &heeof stereoisomers thereof derivatives there, nalogs thereof, and combinations thereoT 2~ Nin iitrting eapies of caner vaccines' useti in te preseannventionainclude 30 A A from Actve Biotect DO A immNorthwest BoteampetosE I from 1DM Phatma. GV 1001 snPhanne n4205 5 noar klar Pharmacenticais. INC6 22$ tnntrogen l'herapeultics and Stimuvexan onflionrkere 60 lExalp]s of idiodn ripevts ittar tlimUe t i such as 4 Se-*tdu~ CQ 'Sr R 3 y 5 y 90 O~ 31g 19 mSr Pt S S t21 Ten-Emtiag exanple ofsga tramnduden molediaand patas that aybe Pathw 1wn Patwiy 2&b V Pospo Shn h "" n T -- ----- -------- -------- n .......... ay~ Pthway 4 rt392 ph His, ssh2 t~-- ----------- -o -- ------ --------- -- P t h w a y 5 - b--- s p- - - - - - - - - -- - - Pthwas6 - 1jD4 $ e 1 Toho4' z* rBsh' _________ Thethwiiy 7 3'GNR3 GF3-lRhzispfa Sn' 353L P3Q 31K 3r5 SI -- --- -- -, ~ --- --- -- .1 33-3 -- --- ----- --- - . Patway D, 4E IN_ _pha Pathway 19 K-0T 2 R 2ithosph___ Panhway 1 33-ln t.3-hmspho ________ ........... ---- r-------------b ?adtwy 1P p__o Pathway 12 VMa5rR 2 3 i PaThway - - --------- R-- Ph - - - _ _ _ 3- - - - - - -_ Pathway l2 Okla ranhway4 1 16 vaara acc ee sre - ----- Pathw 2' VEG-ID P___Y Si__ ----- --- -P 2- Pahaywoo, 3 WO Pathway X4 TE2 _ _P A - Pathway rPathway 2 FR P bphEPr -- t--way 39 -- - ... ----------- ---- _ Vasbway 341 Pa--hway 32 p---- P--p-- -- T---3 . t.p...T.t..R . ...... - - - - - - - ------- P h-r - -- h- Paibway 34 Phtspha (51)cmp P- 03K (ah Akt~ iAS Gtn ~ 5 ) (s (P(22) GE% (3) I -- 4~ ------ ......... -- -is' -' ---- ------------ k *---------- - ----- -~ - --- -------- ----- .......... ~>'-~- r - --- - - - ------------ - &1 ) - -------- --- w s ------- ------- ~ ---- Pathway S ?"67 thp s39TO f0215] Non-litting examples of signal iransduction mol~culehs that can he interrogated int a coeulr e:Gac include. without lhmitaion, receptor tyrosine khnases no-ecpor tyrosine khaaes, tyrosine .kinas-e signing cascade componeni. nuclear hormnone receptor, nuclear 5 receptOr con gators. nuclear receptor reressors. nid combiotmoo thereot In certain insltces, the plurality of signal transduction meculeos is, selected hom the roup comistmg of EGFR (=rb01), RER (ErbB2) p95HER2, HERS (ErhB3- RE4 (ErbB4), P13K. SH-KC Ra. SRC MEK, NfkBdkB mTOR, PK, VSGF, VEGFR I V.EGFR2 VEG1R3, r EPTA, EPM-BS EPH-C, EPH-D, c-MET, PPR, c-KIT, FT-3 TIE-1, l-2 c-FMS, POGRA, 10 PDGFRB, Ab, FTi 3, RET, HGR, FCFR . FT R2 5FF3, FG5R4, 1GF-1R ER PR. NOR,. A )BL AKQT, ERK12 (MAPKERK (MAPKC > ,~ PDK ysl PD1K2, PTIIN4, SOK13. 43 BP. P78611, protein yrosine phosphatases (eg. PT , PTPN3 BDPT 11 e, receptor diners, (GSK'-2p, P1P2. PP3, p2.7, -ad cszmbintations tieseof In particular embodints,. the one or aore signal transduction molecuKes coprses Erh l HR. ErbBI2HER2 5 p9511ER2 1 EbB3/HER3 c-MET OPL c-KIT, P2DK SUC, VETR, or donations thereof, {t2161 Total cyOpSSond activation phosphoryiatie)geve andoratos can he detemimed usin ny f vytietyof teclmiqu ertam enboanen the oxplnn andkor atitvaion (og, phosphoylat on) ievel and/or sam f signal tratnsdudon cu in FNA sampUe is detected wth an ia assay such as asinle detection as or a roximity dusideteroassay(g.a CQllaboateetronIty .mmoissay (CONP4Y}as desibed herein [27 in particular it psce f acti sn rransducon molecules is detected using anhximmunoassay comprising; (i) incubatitg a cihAr extract of EN A cells w aI a pIuraliY of dimon series of caporare iantibodies specific for the one or more signal transducticaimolecules to form a plurality of' captured signal transduction molcues, wherein the 10capture anibodies are restrain on a sAid support; (ii) inubating the plurality of captured signal transduction muolecules with a. plural ity of det leion antibodies comprising activation statendependent antib~oies and a phurality of activation statt-dependent antibodies specific for the corresponding signal transduction molecules to tem a plrality of I S ~ detectable captured si goal ttansdutctiona molecules. wherein the activation state-inrdependent antibodies are labeled with a facilitating noiewt the activation state-dependent anibodies are labeled with a first member of a signal amplification pair, and the a t moiety generates an oxidizing ageit which channels to and reacts with the first member of the 20 ~ signal amiplificanor pair; incubatliAge urality of detectamhi captured signaltamsduction molecules will second inembe of te signAl amplifiction paIto generate an amplified sigal; arnd (V) detecting the anphfied signal generated orm the first and second niembera of 5 ~~theagal eamidifitikm pai. f12181 in certain instances the method May further comprise the step (e) of pniding the result of the comnparson obtained in step (d to a user (e~g, a clinician sueh as an oncologist or a general practitioner) in a readable format, In some instances, the method may furtther comprise ending or reporing the result of the compaison obtained in step (d) to a clnician. .0 egg, anr oncologist or a gneral practioner. in other instances, the method may further comraise recording or storing the result of the conparison obtained in step (dI) in a computer dataase or other suitable machine or device for storing information, eg, at a laboratory. 63 BV lreast Cancer {0219| Breast cancer is the fifth most common cause of cancer death worldwide, a:et ung cancer. stomach cancer, liver cancer, and colon cancer, In 2005, brast cancer caused 50200 deaths worldwide .Aong women world ide, breast ancer i the iost common 5 cause of cacer deatI i the United States. breast cancer is the third most common cause of cancer death, aferhlung cancer and colkn cancer. in 2007, breast cancer caused over 40,000 deaths in the U. Among women in the US., brest cancer is dhe most conmn cancer and the seandmost common cause of cancer death In fact, women> t lifetirne chance of developing bnvasivre breast cancer and a I in 33 chance of breast cancer S0 causing their death, Th minber ofcases of'breat cancer worldwide has signicant1y ncrmased since the 1970s, a phCn non partly blamed on modem lifestyle in the Wetern worid, B cause the breast is composed of idenntica tissues o nades and fnales, breast cancer also occurs in males. though it is less common, {51122W Breast cancers can be deseribed using for~ different classifientidon schemes ec based Oildhe .fdolniuacrhtlia 1 Patology, t hpaogi can categorize each tumor based on its histological appearance and other crtri The most common patologie ypes of breast cancer arc invasive ductal carcnoma and invasive lobular carcitnm. 20 2 Grade of tuMnor The hisrtoical grade can he determined by tho pathoogist under a microascope, A welil-dtferent.iated (ow g radio) tumor resembles nrald isue ApCory differentiated (high grade)\ tumor is composed of disorgaunrdm cells and does not look like normal tissue. MKoderately differentiated (intermedi ate grdltumtors are sumewhere in between, 2$ Pron and gene expressionvstatus. Braest cancers can be tested for c version and/or activation of signal amnsduction molules such as, for example, the estrogen receptor (ER), progesterone receptor (PR), and HER2/NeumEb132 As described herein, the profihe of expression of a given tumor aids in the prediction of its prognosis and assists the oncologist in sdeetmg the most i apopat 0 treatment 4.Stage of the tumor Breast aners can be staged accordingto the INM (assif etica sy5X~fl -. Tamor. Five values (is TI 12, T3 or T4) depending on the presence or absence of invasive cancer, the dimensions of the iasv cancer, and the 5 presenc or absence of invsion outside of the brast feg, to the skin, of' the breast or to the musclie or ribcage tmderneath}, b. Lymph Nd, Four' values (N, N1, N'2, or N43) depending on the numberQ size, and location of metastaic deposits in lymph nodes. . Metasdnse. Two vahnes (MO or MI ) depending on the presence or absence of metastases oter than lymph nodes (so called distat metastaSes, *g, to o brain, lung, etc), Padmi gne j0221j W/it.respect to pathology, the Wordd~enh Orgmaiatio >scbassiication of breast tumors sets tcrth the foliowing' hitoloia esl 15 1. invasive breast erninonmas such as invasive ducial carciena. (e g;, basal-dike carcinma, mixed type carcirnma, pleomorphic carcinomna, arcinomna wiih osteociastic giant cells. carcinoma with ohoriocarcinomarous nature& carcinoma with melianotic tfeatres),~ invasive lobular carcinoma, tubaar carcinoma, invamive cribriform carcinoma, medOullary Larcinomra, mu cious carcinma and other 20 trnmours willh abundan ucinsr(z mnucinous carcinoma, eymtadenocarcinomat and colunar cell mncinous carcionma, signet ring cell1 carciiinma) neuroendoc i tumours (s g, soid neuroenocrine cariooa (carinoid of the breast), atypical carcinoid tumour, small cell/oat cell carcinoma, lartge cell neuroendloc in camin oma), invasive papillary carcinoma 5 invasive miceropapiary 2 carcinomia, apturire carcinoma, mneaplastie carcinomas (aag mixe~d epilahlillm esenchaymoa mneaplastic carcinomas or pure epithelial mnetaplas tic c.arciomas such as squamous cell carcinoma, adenocarcinoma with spindle cell mectaplzasia adenosquamous carcinoma, and mucoepiderod carcinoma), lipid> oich cancioma,. secretary arcinonia, once ytic carcina, adenoid cystic 3d carcinoma, acinic cell carcinoma, glycogen-rich clear cell carcinoa, sebhaccons carcirnoma, inflammnatorry carcinoma, andi biateral breast carcinoma; 6$ Precursor iesim such as iobular' neoplasia (eg, lobuar carcinoma in situ> intraducta proliferative }esioms (eyg, usual dutal hypepasia, flat opthelial hyperplasia, atypical dauta hyperpasia, da cacnm in situX, micrsinvasive carinomss (a g , central papilcoma, peripherM papilltaa aiypical papilloma, intraduictal popillary carcirinma intracystic papila nin epitheal lesions); 3.&ign epithehal leuens such as lON ding varts (g. sckrosing dtetiosis, apocrinte adenosis, blunt duct adenosis, microgiadular adenosis, adenomayoepithcli adensis). uadial scemplex serosing lesion, and .10 ade. nmas (stg: tubular adenomar, Iactating adeoma pcrine adenoma, pieomorphic adenomi, dnut adenonma); 4. Myoepithelial lesions such as myrce'pitilios is, adlenomnyaepitbelial adenosis, y ithema, and m ali gn'ant myoepiditelinnma; i Mesenchymao tamas such a. sarcoma, haemangiaa aniomatosis 1$ haemangopetcytomta, pseudaaininimatos strcsmas hyperplasia, myo -fibrob lastomia, fibromnatosis (agressive), it uflm&oy myohobn tumour. lipom a : togipoma) graAnslar cell tumor, nuotoiboemna, scvannoma, angiosarcma liposarcoa, rabdomyosareama osteosarcmana loyom anNteweiouarcona; borderline niant i o gde perductal stromal sarcoma d ma olmavt hamtationa; 7Tumors of the nipple such as npple adenoma. syringumatous adenomas. and Pagets disease otfthe tipple 2$5 ~ Malignant yniphus ;9:&eatasaic tumors; and It Tutnoos of thenmale breast sucht as cgynedmstdin albstu or intvasive [Q2 2 Dnctai carcinomani the most commocatype oft breast cancer in women and refein to 30 the development of cacer ells within the ilkdcts of thebreas it comes in tno WinoSm 66 ONvasive ductal carcinoma (TDC), a ivasivc, ulignat oeoplasm; and ducta carcinoma an asie (DOLS), a noninvarive neopiasam, 1DC is the most common form of lavasive breast cancer, It accounts for about 80% of all types of breast cancer. On a mammnography it h usually visualized as a. mass with fe spikes radiating from the edges. O1phydal >eam nations. this lump usadlly feels mutch harder or firmer than benign breast lesions, On itcosici exaomintin, the cancerous cells invade and replacedth surrounding norrnal tissues. DCIS is tenmest common type of nsonihvasive breast cancer in women, As scren amography has become more widespread, DCIS ha beame one f the most commoily diagnosed breast conditias. it is ofien referred to as "tage zer&" breast cancer, W C18 is usually discovered through a mammogramm as very smal spe&ks of cakium known as nmiccalicatons, Howe ver, not all icro c-if ications indicate the presence of'DC1S, which must be confirmed by biopsy. DCIS may be multldoeland treatment is aimed at exci sing all of the abnornma! duct elements, leaving clear margins. After excision tratment oten includes local radiation therapy. With appropriate treatment, DC1$ is unlikely to 15 develop to invasive cancer, Surgical excision with radiation lowers the risk that the DCTS wvill recur or that invasive breast cancer wrill develop, [02231 Invasive lobular carcinoma (LC) is a type of breast cancer that starts in a lobule and spreads to surrounding breast tissue. If not treated at an cadystage, ILC can move into other parts of the body'. ubb as the uterus or ovaries, LC is the second most common type of 20 invasive breast cancer, accounit for about 10-0 5% of all breast cancer cases. ILC is characterized by a general thickening of an area of the breast, usually the section abeve the nipplek and toward the atm, ILC is less lUkely to appear on a nmanmmogram,. When it does appear, it may show as a mass with fine spikes radiating frm the edges or appear as an asynmnetry compared to the other breast. 25 T~rfe UiZ24I A number of alterations in key signsal transduction compoents have bee-n demonstrated in bregst cancer. These include: h ER irutations that result activation; acsiatien of other receptor tyrosine kinases such as c~ME I;ER activation with E2 and HER3 activation or HER! amplfcation; ECFR activation with PDK mutation; EGF? 30 acuation with PTEN deletion: and EFR actaaion with Ras mutation. Various alterations un different components of sial transduction pathways have been targeted by various forms of chemotherapy 67 ifl2251 A t the samne tome, the tormation of new blood vessels to tumor cells> a process tenned angiogenesis can be targeted. VEGF is an endothelial cel survival factor which is essential. for .tnnation otf new blood vessels. Accordingly, one approach to the mnodulaetion of VEGhnmediated angiogeess is to use antibodies directed against the VEGF potein itself or VEGFR Bevacizumab, atrecominant humanized monoclonal antibody to VEGF. ants synargtstcally with chemotherapy and has been shown to improve survival i patients with comZlorca, breast and lung cancers 102261 All endocrine therapies are designed to block strogen receptor (ER) tuwetion in a uique way. for example, selective~ estrogen receptor modulators (SERMs) roeb as a tamoxifhen bind ER and partialy block. As activity, Ovarian ablation, inteinizing hunnone releasing hannone aonists and aromatasc inhibitors such as anastrozole (A iIdex"), let rozo Ie (Femat) an xeetane (Aromasin') reduce the level of estrogen and inhibit ligand-induced activation of ER, The idead 5ERPM should function as an a xt-estrogen in the breast anrd utemus and a partial estrogen agonist in the skeletal, cardiovascular. and cental IS nervous systems, as well as the gastrointestinal tract arnd vagtina., [0227j Stemidal ani-estrogens such as fldvestrant bind BR more tightly, hence completely blocking ts function and inducing receptor degradation. [)22$] Thancxifen, a selective estrgen meep tER) mnodulator, is the most widely used drug for the treatment of ER-positive breast cancer. Ad nant therapy studies of ramostren 20 show a 40% to 50% redluction in. the odds of reurrence and mortality, Tanmoxifen also provdes temporary remission in 30% to 50% ofpatents wih metastatic disease, and it is also effective in the prevention of breast cancer, 102291 Aromatase inhibitors ate: becoming tihe standard of' care in the treatment ot postmenopausal women with breast ancer, while tai fen remains theandard in 25 premenopausal women. Although ammatase inhibitors may be slightly more ffectve than tamoxifen, it mmains an important drug because of its documented benefits in sequence with these agents for adjuva. therapy, and because it will continue to have a role in metastatic disease. :0 102311Dr ia (noesponse to initial therapyprinmary reistane) and acquired resistance dease nlapse or progressiOn ater showxga initial responseto therapy; secn resistance) to tamnoxifen are ma jor probkems, As a result, understanrdingt tumor biooy andi the mechans of resistance may provide significant clinical Pimpiions. (0I23I] ER/PR biology: ER and PR are nuclear hormone receptors which function as tran ion fctor in the nucleus whe n they are bound to ligand(siER and PR have 5 similar structures and contain, a DNA bhnding domain, a. dimueriation domain, a hornone binding doman, and several transcrpua activating domains. A grater reduction in ris fr recurrencee was noted foxr paisens with ER positive, PR positive tummors compared with those with KR positive PR negative tunce e0232J FR fuciou o Htorone binding to ER activates the protein though r phosphorylation, dissociates chaperone proteas such as hea-shock protein 90, and alters its conformation, One bound ("ivated") ER. then dimrerzes with another receptor. and the dimer binds to estrogen response elements (specitie DN A sequences) reen in the promoter of astrogen-respon~sive genes. Promoter-bound ER diners form~ a comnpiex with cXf~ regulatory protein c n amplifbied in breast cancer I (AUI or SiRC3) that coordinately act iS to influence the transcription of estrogen responsive genes, Typically, co-activators bind ER when. the receptor is hound by estrogen, while co-repressors bind when ER is beon by tamnoxifen, AEB1 is ovr~xpressed in 65% of breast cancers and the corresponding gene is ampied in 5%, igh levels of AfIB1 way contribute to SERM resistance by enhning estrogen agons activiy (e.g, treat with aromatase hiitorsi). ER imers also form 20 caioplexes with corepressot proteins such as NIOR to downrelate gene expression of certain genes (0,1 HOX13), (0233j Several kinaises in the growth factor signaling networtks can adso activate Ei in a process termed liganidependent activation, Under certain conditions such as high IbB amilY activity V eg high IER? or HERi activity). ER bound to tamexifen com;pleAs with 2.5 ATEB1, resultng~ in increased estrogen agonist activity of tamnosi fen (e g., treat with fulvestrant or asonmatase inhibitors alone with kintase inhibitors), [9234] This nemnucoleer ER action or mtembrane-hsitiated steroid signaling (MIS) $a ccurs within minutes of the addition of estrogen, SERMs such as tae ifen may also activate rnmrane ER. These receptors have been found in bone, neural, uterine, It, and endthelial 30cells,. Mechanisms by whieb estrogen activates membrane ER function are beginning to be clari fied. Direct interactions between ER with a variety of mecmbrane-signaling molecules such as the insulinmike growth factor I receptor, the P85 regulatory subunit of PK, Sr, and 69 She, a protein which may directly couple ER to a variety of growth factor tyrosioe kinase receptors, have been observed. Acti vation of these pathways by estrogen sends powerful cell survival and cell proliferative signals via activation of AKT und MAPK hn additloa. these inases can phosphoryhate ER and its crgulators to augment nuclear ER $inaing, $ Phosphorylation of these proteins can also increase the estrogen agns-ieactivity of amafen and other SERandsi 5 02351 The pure ani-estrogen thivestrsni does not activate mmrane IR in this way; hwaer, SERMs such as tamoxifen do activate membrae ER in a manner similar to estrogen, The membrane effects offER. like its nuclear acivty ma eceleeptor 10 subtype, and ligantd-speiic. and it may also be influenced by the growdh factor dialing milea being muchmor trmi ent lance, inx. breast cancers overexpresigEbi HER2, Stimulation of the MISS activity of BR by tamoxifen and other SERMs may, in part, explam the resistance to these agnats sometimes observed in HER2-overexpressing tumors, 102361 In addition to ER XWmenons assisted with. the nuclus and plasma membrane (membrane-initiated steroid sigoaling; M)SS, ER conjugates with other pathway molecules to facilitate subsequent tumor progression. This moliecular crose-talk. can best ho treated with aromnatase nettitors and no3t SiEMs 102371 ER has at least twa major frictions. It serves as a trarpion factor for estrogen regulated genes and a co-activator for other transcription factors in the nucleus. It also 20 functions in the cytoplasm and in the plasma metnbne to activate growth teetor signaling. In some breast tunors, particularly those with hily active growth factor signaling pathways such as ER2 atuplticeation, a vicious cycle is established in which estrogen activates growth lictor signing, arnd tre growth a-tor signalhtg pathway farther activates ER, Estrogen in such tumors wOuld be expected to be a domant factor by activaing mult pathways 25 important in or progression, This molecular crosstalk has importam implications for the treatment of breast cancer. As an example, estrogen-deprivation therapy with aromatase inhibitors should be more effective than SERNs to HER2 amplified tumors by shutting off both the inuclear-<bnitiated steroid signaling and MISS activities of ER Metasti Disease 30 0*23$] Two-thirds or more of breast ouors are dependeton estrogen for growad A mn-er of esimogenkblocking agents may be usactfitieannont ofmnetastatie reastcatcer. The treatment response to these agents is unpreditable~ howaeeand approximatelyone botb0 third af patintsith i etastatic breast eacer yih receptois tot Stestgeorprongstrne uo beneo iom hormonal therapy Neady all patientsind metastatic breast cancer will eventually become resisnet to hoonal therapy despite te fauthat tie hormone receptor are stil ptesent 5 {0239] Therapyeeedon is deternned based on actitron t g iag p ys or better understanding ano a g iipat ca embod , h present wn advaiageously provider an.assay nedhodology along with a diagnasti.c/prognostic chip to hep nelcitsdeid tebes;t ratemhrindiidlpatients nt oitruetion of Antibody Ara ys 10 1024f] in certain aspects, the expression level and/or activation state of one or mnore (eg phnraiity) of signal transduction molecules (e~~g: HER2 signaling pathway components) in a ceihdlar extract of tumor cells sa as breast cancer cells is deleted using an. anibody-ased array comprising a dilution series of capturre antibodies restrained on a solid support The arrays typically comprise a plurality of different capture anibodies a a moge of capture 15 and body concentrations that are coupled to the suitfae of the solid support in dtifttrenat addressab locations, in one pmartclr eshadiient, the resent invntonprovides 'an adresaleara .haavng superidyn r n ig a prluity of dilution series f capture antibodies restrained on a soWi support, in which thmecaprure annbeodies in each ditutiorn series are 20 speWcC for one or more analysts corresponig to a. component ota sinal tmnsduon pathway and other target protein, ln various aspects, this embodiment inrchides arays that comprise componetO of signal transducion pathways characteristic of panicular tumors, mg, signal transduction pathways active in breast m cei!s (gt ILER2 path way The the irwnaion may be advantageously practiced where each signal tasduction molecule or 25 other protein of interest with a potential expression or activation defet causing cancer is represented on a single aray or chip. in some aspects, the components or a given signal transduction pathway active in a particular baumor ell are arrayed in a. linear seq uece that cormesponds to the sequence in which intorrmation is relayed through, a signal transduction pathway' witi a celt. Examples of such arrays are described herein. and also shown in 30 Figures S~9 ofPCT Publicaton No. WO2009/108637 the disclosure of which is herein incorporated by referne in its entirety fo all purposes The capture antbodies speciic for 71 one or mo~re components of a gie sga trasdution pathway active in a particular tumor cll can also be: printed in a randomrized fashion to moinimizxany vsurface-reated artifats, {O2421 Th. e solid support can comprse any suitable sutrac or immZobilizing protem. Examples ofsoiid suppors include, but are not bmited to, ga a glass slidto). pbtstic chips, pins, filte beads, paper, membranes, ftber bundles. gels, etal, ceamics and he like. Mlembranes such nylon (Biotranat )CN Bliomnedicals, nc, (Costa Mesa, CA); Zeta Probe* Bio-Rad Laboratories (H~ercules, CA)), nitroelulose {Protrant, Whattman Inc,. (Florham Park, NJ))and PVDF (mmRobilon" Mlipore Corp. (Dillerica MA)) are suitable fOr nse as solid supports in the arrays of t present invention, Preferbly, the capture lo antibodies are restrained on glass sides coated with a nitrocelulose pobymer, ey FAST Sliidcs, which are commercially available frm Whatman Inc. (Florhamt Park, NJ). [02431 Particular aspects of the solid support which are desirable include the ability to bind large amounts of capture antibodies and the ability to oind capture antibodies with mxinitnad denaturation, Another suitable aspect is that the solid support displays mnial wickingg" 1. when antibody solutions c'ontaining capture anrtbodies are applied to the support, A solid support with mrdinial .ci allows salaliu oon applied to the support to result in smltl defined spots oa immobilized captu antibody [0244) The capture antibodies are typically directly or indirectly (eg via capture tags) restrained on the solid support via, covalent or nonecovalent intemections (e~g ionic bonds, 20 hydrophobic interactions, hnydorcgen bonds, Van der' Waals forces, dipole-dipole bonds) lIn some embodiments the captate antibodies arne cavalently attached to the solid support using a homobifuncctional o beterobifctittonal croslnker using standard crosslinking methods and contditions. Suitable crosslinkers are~' commeriy availablenufrm vendors uc cg Pierce Bliotechnology (Rockford. i1t 2$ I0245] Methods for enerating arrays suitable for use in the present invention include, but are not linited to, any technique used to construct protein or nucleico acid arrays, in some enixodimnents, the capture antibodies are spotted onto an array using a microspotter. which are typically robotic printers equipped with spirt pins, blunt pins, or ink jet printing, Suitable robotic systems for printing the antibody arrays described herein include the PixSys $000 3~0 robot (Cartesian Technologies; hrvine, CA) with ChipMaker2 split pins (TrleChcrrm international; Surnyvale< CA) as well as other robotic printers available from BlioRobics JQQ Il 0"aw (Wobun, MA) and Packard Tnstrument Co(iexiden, CT), Preferbly at least 2, , 4. 5, or 6 repies of eah iatry artJxxly dilution are spotted onto the array. oefete citqwu eachA' captures '~ocx [O246I Another method for generating arrays suitable for use i te present invention comnprises dispensing a known volume of a capture antibody dilution at each selected array 5 position by cumtacting capillary dispenser onto a solid suppont under conditions etivte to draw a defined volume of liquid onto the support whern this process is repeated using selected capture antibody dilutions at each selected array position to create a complete arrat The method may be practiced in forming a puaty o arrays, where the solutin~ depositing step is applied to a selectedpostion on each of a plurality of solid supports at each 10 repeat cycle. AIfurther description of such a method can be found, eg ini U. Patent No, 5,807,522. rY2471 h certain instaIeIaInies fonting onWS ppet can be u 1d tognc ethe: artibody arrays Poreample, he desieapt anbody dilution can be loaded ito the printhead of a deAtop jet tter and printed ontoa suitable solid suppt see, eg e@ 15 aCeL. CtW ,-hem. 44:20362043 (1998) (02481 in some embodiments, the army generated on t sosold support has a density of at leat about 5 spot/cmn and preferably at least about 10, 2 30, 40, $0, 60 70, 80 1 90, 100 110, 120 130, 140, 150, 1 60, 170, 180, 190, 200, 210,220, 230, 250,275 30A 325, 350, 35,400, 423, 450., 47$, 500. 50, 60 650, 700, 750, 00, 850, 900, 950, 1000,2000 3000 20 4000, 5000, 000, 7000, 8090 or 9000, or 10,000 spots/t? 112491 hI ceraim instances, the spo t s on the solid support cach represents a different cape antibody. in Cenain other instances, muiple spots on the soiad support represent the sane capture antibody, ag, as a dilution series compisig a. series of descendg capture an:tibody concetatations. 25 041 f} Additional exanmples of methods for preparing and aostracting atbody array; on solid suppose aredesdcbed in AS. Paten'os. 09599 77T239 638082 097,73, 117953 8.d 972 S Patent Pu,2caion o 2006')58.0 2006026383 20060t9268O, rad 20070054326 and4 aan'm et atMethode Mo?, Riot,6 20:042 (004 30 {$51 Medhods far scanning antibody atwas are knowna in the art and inclosde, without lniaiomany technique used to scan protein or nucdeic acd arrays. Microarmy scamne 30 V7.3 ftl for use te present nventionare available redrinilhnet (Bostot MA) Agilent Technologies (P an Alkt. A), Appi.jed Precision (bssaqnah. WVA) 6S lumnizs tic (iierica, MAIand .tzolustruments (Union iy CAOO a ndiing ale a G51 StatAnrav3000 foiflutorescence detection can beused with iinaene soware for (0252 in some embodmnens. the essay for detecting the expression end/or activation level of a particular analyze (tg. a signal transduction molecule such as a component of the HER2 signaling pathway) of finest in a~ cellular extrc of cls suh as tumor cesls is a multiplex hi.bgh-4hroughpmu two~antibody assay having superior dynamic range. As a lnn-limiting example. thwo antibodies used in the assay can comprise: (1) a capture antibody speedier for the analyte; and (2) a detecton antibody specific for an activated form of the analyte i.e. ativaton stateependet antibody. bThe activation stadependemt antibody is capable of detecting for example, thme pltosphorydation, ubgination. and/or complexation state of the I m analyte, Alternatively, the detection anmibody comprises an activation state-independent antibody, which detects the total amount of the analyte in the cellular exact. 02513] in one particular emboddment, the two-antibody assay for detecting the expression or activation level of an analyte of intrest~ comnprises: (i) incubating thme cellular extract with one or a piuridity of dilution series of 20 capture atibodies to form a plurality of captured anatlytes; (ii) incubating the pluality of captured analytes with detection antibodies specific for the corresponding analytes to form a phurality of detectable catu~red anaiytes, wherein the detection anibp&dies comprise acivation state-dependent antibodies for detecting thme activation (e~g., phiosph~oryatiomn) level of tihe 25 anaiyte or activation sate*-independe~nt antibodies for detecting the expression level (e g, total amnoun) of the analyze; (ini) icubating the plurality of deteetable captured analyses with first and second members of' a signal amplification pair to generate an amplified signal; andA (iv) detecting the amnplified signal generated froma the first and second mnembter of 3(U the signal amnpification pair. 102541 The twoeantbody assays described herei are typically antibodybased arrays which a nmprmse a piuraliy of different capture atbodies at a range cicapture antibodiy QecettadonsO~that are coupled to the surface of a slid support in different addressable lesos Exaegefsuitable sold supportONfor use in the prset inveution are described fO255 The capture antihadiaes and dietootion antibodies are preferahly selected to minimnize S compen between the. with respect to ansite binding both capture and derecton antibodies can sininltaneoudlv bind their conesponding sigoh transducion reeal ee) [02561 In one embodiment, the detection antibodies compOse a rt member of a binding pair (eug. hiotin) and the Arst member Of the signal amplification pair composes a second mrember of the bindn pair (eag;,streptawidih The binding pair msenbers can be coupled 10 directly or indirectly to thre dtection antibodies or to the first member of the signal ampl ication pair using methods wellknown.' the art. In certain instances, the frst meee of the stgnal ampliteatio pair is a pexidase (ag. horseradish peroxidase ('RPa estahne chioroperoxidase. cytochrome c peroxidase, ensinophit peroxidase, glutthione peroxidase, lactoperoxldse, miyeloperoxidase, thyroid peroxidase, deilcdinase. ec), and (t second member of the signal amplitcanon pair is a tyramide reagent (eg, blotinayramide). in these itancea, the amIplied signal is generated by peroxidase oxitnn of the tyramide reagent to produce an activated tyramside int the presence of hydrogen peroxide. (H2G), [2571 The activated tyrarmide is either directly detected or detected upon the addition of a signatietecting reagent such as, for example, a saeptavidindabeled flurowphom or a 21 cobination of a streptavidinlabeled peroxidase and a chromogenic reaemn Exmples of fluropirs suitable for use in the present invention include, hut arc not b noted to, an Alexa fluor dye (.. Alexa. Fluo* $5S), fluorescein, tRuorescein isothiocyanate (FITC), Oregon Green "4 rhodamine, Texas red, tetrarbodamine isathiocynate {TFJTC)\ a Cy Dyves fhuor (e g, Cy2, Cy3, CyS). and the like. The strepavidin labe can be pled dectly or 5 uiniretly to the fuorophore or peroxidase uing methods wettmaown in rhe at NiO limiting examples of chromnogenic teagenta suitable for use in the present invention include 3', 5 ,SXieramethylbenzidine (TMBt), .33 M-iaminobenzidine (DAl3 22 2-%zina-bis(3 ethylbenzothiazoline6sultonic acid) (ABT'S), 4-chlorotnapthol (4CN), and/or porphybnogen. 30 0258 An exemplary protocol f performing the two anybody assays described heen i povide _ Eample 3 of PCTI Puhleation No, /02009/10g3he diselosuteof which is herein incorporated by enc initsentetty r allpurposes 5$ [0259 in another embodicentt a. antibody appn he present invetion proxies a method for detecting the epression or activation leel of a tiamated eceorr he methd p tWping (0 incubatng the oedliar extract w a piofWalit head specifid far an extraedthek domain (ECD) inding region of a fndlength reeptor ('1 remomg t p ray of beads frihe cellular e>tmot hereby reming the fuidlengtinreceptor teo oi a celhuar extract devoid of the htidength (n nicabnating the celhdar extract devoid of the fou-dength receptor with adilution 10 sei~esof one or a plurality of capture antibodies speciic frn an intraart domain (101))inding region of the Mu-length receptor to fano a plurality of captured truncated receptors; )lv incubating the plurality of captured truncated receptors with detectionr antibodies specific fon an ICD binding region of the flngth receptor to mfon a pWeity of detectable captured truncated receptors, wherein the detection antibodies comprie activation state-depen dent antibodies for detecting the acti-ation (rtg, phosphozylation) level of the tnuc-ated receptor or ativati on. ttre-independent antibodies for detecting thc expression levei (ag, told amount) of the truncated receptor; inubtigheplm!ty' of detectable cptured tncte ecprs with first aind second meenbtes of a signal amp- icarion pair to generate an amnpliid signal; and (vi detecting an amplified sige generated from the first and second members of the signal. arinfllation pair, 2$ [02601 i. certain enbodimnems, the intncated receptor is p93HER and the fullngth receptor is HE1R2, In certain other embodrnents, the plurality of beads specific for an extracellar donain (EC1) binding regain comprises a streptavidibitm par whterein the biotin is attached to the bead ad the biotin is attached to an antibody (eg. wherein the antibody is specitc for the CD binding region of the full-ength teceptor). 30 10241 Q Fi 14A of PCT Publication No. WO2009/108637, the disclosure of which is herein incormred by reference in its entirety for aft poses, shows that beads coated with an antibody diected to the extracelauhr domain (ECD) of a receptor of interest binds the ful legh eetot (eg, H-ER2). but not the truncated receptor (eg~, p95H.ER2) to re-move any length weepp 76t llidength receptor from the asa F'iur 14B of PCT Publication No, W02009/108637 shows that the tru cated receptor (a l, p93"1ER2 once bound to a capture antd ay then be detected by a detectin atibody that is specifi fourth intraceihdUax domn0 (ICD) of the felldength receptor (eg HER2). the detection antibody may be directly ennjuted t h .scradtsh perxidase (HRP). Tyramide signa amplification (TSA) may then be erformed to generate a signal to be detected. The exson level or activation state of the trncated receptor (g, p95HR2) can be interrogated to determine etg, its total concentnalm o ts phosphurylation state, ubiquitation state, and/or comrplextion state, [06 hI another otmbodimvent, the present svntion provides kits tor performing the two~ 10antibody assays described above comprising: (a) a dilution series of one or a plurality of capture artibodies restrained on a solid support and (b) one or a plurality of detection atibodies (ag, activaion stateside dnt antibodies and/ornativation state-depedtt amibodies), In some instances, the kits can starter contain instructions for niethods of using the it to detect the apression evels and/or activation states of one or a plurality of signal 1 transduction molecules of cls su as tumor cells. The kits may also contain any of the atdiioal reagents described above wihh aspect to performing the specific methods of the present invention sucha as, for exrample, first and second members of the signal amplification pair.tysaande sigdalamplitietion reagents. ashhuffers, tc. VlL Prrimity Dual Detection Asays [20 12631 In some embcdiments, the assay for detecting the expression and/or activation al of particular analyte (atg a s gnal transduction molecule such as a component of the HER2 signaling pat hway) of interest in a cellular extract of cells such as tumor els is a muipe. higTh-throughput proximity (f&, three-antibody) assay having superior dynamic range, As a nondmiiting eUmple, the three antibodies used i the proximity assay can comprise: (I) a 25 capare antibody specific for the analyte; (2) a detection antibody specific for an activated forn of the anralyte (i activation ste-dependent antibody); and (3) a detection atibody whieb detects the total amount of the analyte ("e. activation state-idependent antibody). The activation state-depenident antibody is capable of detecting, e.g. the phosphory lation, ubxquitination, and/or complexation state of the analyte, The activation state-iudependent 30 antibody is capable of detecting the total amount of the anayte, 264 o particularebodimntthe provinuty asay for detectingonte atvation lev or status of an analyte of interest comprises (i) incubating the celhdar extract with one or a phurabity of diluion series of capture antibodiies to form a plurality of captured analyses; (ii) incubating the plurality of captured atnalytes wit detection antibodies comprng one or a plurality of activation state-idependent amnibodies nd one or a plurality of activation statedependent antibodies p corresponding anytes to form a pality of detectable captured analyses, wherein the activation statendependent antibodies are labeled with a faidlitating moiety, the activation saa dependent antibodies are labeled with a firt member of a signal amplifiation pair, and the: fcilitating moiety generates an 10 oxdzn agenm which channels to andi reacts with, the first member of the signal aimpiliation pair; (iii inuating the pluiy of detetalble captured analytes with ac ndm be of the signal amnplification pair to generate an amplified signal; and (iv) detecting the amuplified aigtd generated fiem the first and second memnbers of pats. the sign al apfictin pa.o t265 in another particular emrbodiment, the proximity assay for detecting the ativation level or status o an nayte of interest that is a truncated receptor comprises: (f) incubating the cellular extract with a plurality of beads specific fr an extracellular domain (ECD)) binding region of a fid!~length receptor; 20 (ii) removing the phtality of beads from the cellular extract, thereby removing the fiu-length receptor to fon a cellular extract devoid of the fillength receptoo (ii) incubating the cellular extract devoid of the fudbength receptor with one or a plurality ofcaptwe antibodies spefi is an intracellular domain (iCD) binding region of the ftuhlkngth receptor to forr a plurality of captured truncated receptors; (iv) incubating the plurality oa r trunated receptors with detection antiodis copriing ne r plurality of activation state-ind~ependent antibodies and one or a plurality of actvation sate-dependeat antibodies 30specific for an lCD binding region of the fidength receptoxr to fann a plunliy of detectale captured troncated receptors, wherein the activation sta te-ndependent antibodies are labeled with a facilitating moiety, the activation stategependent antibodies are labeled with a tist member ofa sitra rem~iicaia pdend'the falhtin oiety genertes at oxidizdng age n c hannate and rats with the trst member ot' t sna aopfitaton patta (v) acaubaita uraity of dotetaba e aptured trnced eeeptmetVn1lit scondnemberotche siga ampfication pail to generate an mpbied signal au detectingg the amrpiified sign generated frontd te fist a second amndso the signaIamplincotion pair. [02466 in certain embodiments, the truncated receptor is p95F1ER2 and the fullength 10 receptor ~s HERR2. I certain other embodiments, the plurality of beads specific for ant e>:tracelliar domain (ECD} binding region comprises a streptavidin-bioti ir h thle biobtn is autached to the bead and the biot in. is atached to an antibady (eg whcern the ntxibcdy is specifle fan the ECD) binding region of the full-length receptor). [061It alteirntive ebodimsents. the activation stnAasependent antibodies can be Pabeied withh a faciitatig moirty and the activation stae-independent antibosdies can be hdbeled with a fir. member of a signal amplification pair. IO268)t As another non-limnitin examle, the three anti bodies used in the proximity assay can comprise: (1): a. capture antibody specific tot the analyte; (2) a first detection antidy specific which detects the tal amount f tanalyte a t o statendepndent 20 antibody}; and (3) a second detection antibody which detects the otal amount of the analyze (Le a second activaion. sate-independent antibody), in prerred embodimems, the first and second activation stateindependent antibodies recognize different (esg distinct) epitopes on the analyte O269) In one particar embodiment the proximiy assay for detecting the exprssior level 2 of an analyte of interest comprises: i)s incubating the cellular extract with one or a plundity of dilution series of capture antibodies to form a plurality of captured aral ytes; (ii) incabating the plurality of captured analytes with detection antibodies comprising one or a pluraluty of first and second activation state-independemr 30 antibodies specific for the corresponding analysts to forn a plurality of detectable captured analics. 79 wvherein the first activation~ state-independent antibodies are labeled with a facilitating mnoicty, the second acivation State idpendtent antihodie are labeled swit; a first member of a signal amplificanofl pair, and the facilitating moiety generates an oxidizing agent which chnneis to and reacts with the first 5 mermber of the signal amaplfication pair; (iii) meubnatig the plurality of detectable captured analytes with a second member of the signal amplification pair to generate an ampeblied signal; and (iv) detecting the amplitled signal generated fr-om the first and second nmembers of the signed amplification pair, 10 27&1 In another particular em bodiment, the proximity assay for detecting the expression level of an~ anatyte of interest that is a truneated receptor comprises: (i) incubating the elluar extract with a plurality ot'heads specific tor an extracellular domain (ECDm binding region of a fultlngth receptor; (ii) mmnoving the plurality of beads front the celhular extract, thereby removing the 1 5 fll- length receptor to fom a cellular extract devoid of the: folllength receptor; (iii) incnbating the cellular extract devoid of the fidi-length receptor with one or a. plurality of capture antibodies specific the an intnaielarw domain (LCD) binding reg ion of the full-length receptor to form a plurality of captured truncated receptors; (iv1 incubating the platality of captred trnceated receptors with detection antibodies comprising one or a plurality of first and second activation state independent antibodies specific foir an ICD) hinding ego of the fulidength receptor to fom a pluraidhy of detectable captured truncated receptors, 2 wherein thefir t.ctivtio stae-ndeenen .amide are labeld wih facilitaing moiety, the second activation state-indeprendent anti bodies are labeled with a first menber ofta signal amplification pair. and the tfacilitating moiety generates an oxidizing agent Wich channels to and reacts with the first member of the signal amnplification pair; 30 (v) incubating the plurality of detectable captured. truncated receptors with a second member of the signal amuplithiation pair to gene-rate an amplified ignal; and.

e detetg the ampiied signerated from the firat and second mmbs o the signal amplification pair [0271} in certain embodimrnts the truncated receptor is p95HER2 and the fullrength receptor is HER2. i certain other embodiments, the plurality of beads specific for a extracelluar domain (ICD E) binding region compises a stoptavidintbotin pair, wherein the bioti is attached to the bead and the biotin is attached to an antibody (e.g. wherein the antibody is speci for the ECD binding region of the fdl-lenAth receptor) P12721 in alterative enbodiment, the first activation stateindependent antibodies can be abekd with a fast nmember of a signal amplification pair and the eond activation state I ndependent antodies ca be labeled with at facilitating mietyii ~t273 The proximity assays described he-ren arc typ anie adaaswhh cofmprse one or a phrrlity of dilerent upture antibodies at a range of capture antibody concentratiors diat are coupled tothe surfaes of p solid support ir dileent addressale locations. Examples of auitable solid supports for use in the present invention are described 1$ 7+ above 1(2741 The eaptre antibodies activation statetidependent antibodies and activation state dependent inabodies are preferably sedected to .ni nire comnocti n iheten them Nth TspeCt to analte hiding W alntibodies can simtetaneousty bind tmir responding aigraa transduction makdcules 20 102751 in some embodiments, activation state-independent antibodies for detectng activation levels of one or monre of the analytes or, alternatively, first activation state indepen~dent antibodies fiot detecting expresssan levels of one or more of'the analytes feather comprise a detectable moiety, in such instances, the amount of the detctabie moiety sA correlative 'sthe amouAnt o one or more of the analytes in the cellular exract Examnpks of > detectable moieties include. but are not limited to, fluorescent labels, chemically reactive abelsn enyme labels radioactive labes and the like. Preferably, the detectable moiety a fluorophore such as an Aexa Fluor dy (e~g AiexaFluo 647 fluorescein, fluorescein isethiocyanate (FITC). Oregon Greenm; ilhodamnine, Texas red, tetrarhodanmine isotitocynate (TRITCX a CyDy5 tior (em Cy 2. Cy3, Cy5), and the like, The detectable molety can be 0 coupled directly or indirectly to the activation state-independcnt antabodiea using methods well-knsown in die art 102761 in certain instances activation saae-independen antibodies for detecting activation evels of one or mo e o the anWytes or, alternatively. first actvation ate-independent nitodies for detecting exre son levels of one or more ofthe anaiytes are dc labeled Inoiey ca ith the feeiitatmg moiety Th facilitatng m be cupled to activation state oodepetndent antibodies using methods well-knmown in the at. A suitable facilitating moiety f use tn the present invention includes da moIecule capabug of berating an oxidizing agent which channels to (Ace,. is directed to) andi reaets with. (ite, hinds, is bound by. or iorms a complex with) another molecule in. proximiny (it spatially noer or cose) to the facilitating moiety, Examples oftfacilitating morettos include. without !inion. enzymes such as g ilucotse oxidase or any other enzyme that catalyrzes an. oxtidation/reduction reaction involving molecular oxygen (02) as th election acceptor, and photosensilizers such as ethylene bie, rose bengal, porphyrins, squarate dyes, phihaiocyarnnes, and the like. Non-lunhing examples of axidising agents include hydrogen peroxide (2O) a singlet oxygen, and any other compound that transfers oxge atoms r ais electrons in an oxidation/reduction reaction. i Perabli the presence of a suitable substrate (e. g, glucose 4 light ec.), the facitting moniety (e~g., glucose oxidase, photoscnsitizcx, ek>) generates an oxidizing agent (eg, hydrogen peroxide (H4 %) single oxygen, ae;) which chmmecls to and reacts with. the first mrember of the signal anmpli/inetion ai (e'g horseradish peoi ase(HR haptent protectec by a protecting groupnn an enzimne inaCti vaed by thioethier linkage to an enzymne inhibiitor, 20 ce) hen t two moieties are in mxnity to ea oher [02 77] in certain other instances, activation slate -ndependent anibodies for detecting activation levels of one or acie of. the ainalyes or; aitemaetiveiy trst activation state independent amibodies for detecting expression levels of one or more of the analytes are idiectyaee with the fiacianaoioty i. hyhdzon between. an olgonucleotide 25 linker conjugated to the activauon stat-independent antiodies and a complementary oligonuclentide liker conugte to the taciliating moiety. The oligonucleotide linkers can be coupled to the faihitatin moiety or to the activation state-independent antibodies usinga methods well-krnown in the art In some embod iments. the oligonuclenlide linker conjugated to the flicilitating moiety has 1.00% complementarity to the oiigoinucleotide liker coniugated 30 to the activation stat-independent antibodies, In other embhodiments, the oligonuiclootide linker pai' comprises at leastone, two, thre. tour. live, six, or-more mismnatch rei, a. x, upon hybridization under sizingent hybridization conditions, One skilled in the art will Ow 82appreciate that activation state~independemn antibodies speci fi for different adytes can either be ceonjugated to the same oligonucleotide linker or to different oigoenucleotide linkers. [e2784 The length of the oligonuoleotide lnkers that are conjgaed to the facilitating moety or to the activation siate-independet antibodies can vary. In general, the linker 5 sequence can be at least about 5 10., 5, 20, 25, n A35, 4Q 45, $0, 7$, or 100 ncotides in .kogth TypIcally, random nucleic acid sequence are generated or coupling. As a non lating ex ape. a hihrary of oligo ncle tide linkers can be designed to have three distinct conguous~ dOmin~its: a spcrdmin; signature domain; and congtori domain. Preferably, the oligoncleotide linkers are designed for efficient coupling without destmying , the function of the fC ilitating moiety or activation state-ndependent antibodies to which they ar conjugated {0279 The oligoucotide linker uencs can be designed to revenT or minimirn any secondary structure formnation under a variety of assay conditions, Melting temperatures are typically carefully monitored tot each segment within the linker to allow their participation in 1 the overall assay procedures. Generally, the nwge of melting temperatures of the segment of the tinker sequence is between 1-N0"C Computer algorithms (&y OIGO 6.01 for dtrmini Ag the mAting texnperature, secondary structure, and hairpin structure under defined ionic concentrations can be used to analyze eachi of the three different domalins within cacth linker. The overall combined sequences can also be natyzed for their structural 20 characterization and their nparability to other conjugated cligenucieotid& later sequencs, a.ga whether they will hybridize uder stringet nhyI briizaion conditions to a compemecntlary ohigoneclatiddeiker. [0280) The spaces region of the oigoncleutide Linker provides adequate separat ion of the conjugation domain tfr the oligonucleotide crosslinking site The conjugation domain 25 functions to link nolecuies labeled with a compiememary oligonocientde iinkersequence to the conjugahson domain via ncleic acid. hybridzation. The nucleic acid-tuediatedl hybridization be performed either before or after antibody-anaile {t e antigen) eonmple formation, providing a more 1lexhe assyformat. Unlike many direct antibody cordugathon methods. linking relatively a'll oligonucieoiides to antibodies or other molecules baa 30 mnimsal mpact on the specific athnity of antibodies towards their target analyte or on the finccon of the conjuigated molecules f*2$t}j In somie enmhodimemts, the signature sequence domain of the oligonueleotide linker can be used in complex multp leed potemi assays. MuIdple atiodes can be conjngated wit & gouckeotide linkers with ditfhrent signature sequnces, In multiplex iinmaOssaya, reponrter cligonucleetide sequences labeled with appropriate probes can be used to detect S eross-reactvity between amibodies and their antigens in the multiptex assaytfonnat, 10282] Ojigonucleotide linkers: can be conjugated to antibodies or other molcdeaies using several diP'eren methods Fr example, oligonucleotide liners can be synthesied with a thiol group on either the ' or 3* n The: the group can be depotected using re-duing agents (eFg, TCP-H CIU) and the rsuhing linkers can be purifted by using a destng spin counu, The resulting deprotected oligonucleotide linkers can be cnjugated to the primary amines of antibodies or other types of proteins using heterobifenctional cross linkers such as SMCC, Aernativey, 5 phosphate groups on oligonucleotides can be treated with water schable carbodlibmide HDC to fohrmn phosphate esters and subsequently coupied to samine containg molecules, In certain instances, the di, on the 3 t ribose residue can be ddired 15 to aldebyde groups and then conjugated to the amine groups of antibodies or other types of proteins using. reductive amnination. In certain other instances, the oligoucleotide linker carn be synthesized with a bio i modificaton on either the 3' or Tk end and conjugated to StreptavidinlAabeled molecules. 0283] OQigonucleotid linkers can be synthesitd sing any of a variety of techniques 20 known in the art, such as those described in Usmran et a, ii Aix, Chemt. Se 1 I09:7845 (1937); Searnge ci at YNd A cids Rex, 18:5433 (19$90). Winicott er at Nudt Acids Ret 23:2677~2684 (1995); and Witnot ae dA Methods Mat Bio. 74:I9 (1997). general the synthesis of oligonucleotides makes use of common nuclei acid protecting and coupling groups suth as dimethoxytrityl at the S t end and phosphoramidites at the 3tend. Suihable 2$ reagents tor oligonucleotide synthesis 5 methods for nucleic acid deprotetio, and methods for nucleic acid purification ane known to those of skill in the art, [284j la certain insaoces, activation tate-dependent antibodies Ifo detecting ctivaion levels of one or more of the anualytes or, alternatively 5 second actdvation state-independent antibodies for detecting expression levels of one or more of the analytes are directly labeled 30 with the first member of the signa amplification pair. Thbe signal amplification pair member can be coupled to activaton state-dependent antibodies to detect actnm variOn levels ore activation staredndepe4dent antibodies to detect expression levels using methods weibknor in the art, In certain other instances, acivaion s d dent antibodies or second activation state-independent ant dies are indirectly labeled with the first member of the sitinal amnphifcation pair vita bindinyg between a first metnber of a binding pair cuajgated to the activation state-dependent antibodies or second activation state-independerm antibodies 5> and a second membner ofthe binding pair conjugatedi to the first member of the signal anmpification prThe hing pan embers (gbioti sigtu amplificaion pair member or to the activation stal dependent antibodies or second activation steindependem antibodies using methods well-known in the art Eamples of signal amplificartion pair members include but are not limited to> peroxidases such 1(1 horseradish peroxidase (HRP), catalase, chioroperoxidase. cytochrom c peroxidase, eosinopshtl peroxidase, glutathione peroxidase, Iactoperoxidase, myeiopeiroxidase, thyroid peroxidase, deindinase, and the like. Other examples of signal amplification pir members inchde happens proceed by a pro tcog group and enzymes inactivated by thioether linkage to an enzyme inhibitor. 5 102851 In one example of proximity channeling, the facilitating moiety is glucose oxidase (GOC)) and the first member of the signal am pification pair is horseradish pexidase (HRP) W en the 10 is contacted with a substrate such as ghiose, it generates an oxidizing sgen (ie, hydrogen peroxide (HAOj)). If the HRP is within channeling proximity to the OO, the Il20 generated by the GO is chan e tn complexes with the HRP to fom an 20 20 complex, which, in the presence of the second member of the signal amplification pair (eg, a Chemiiummescent susatmte such as hWAinol or isoluminol or a fluorogenic substrate such as tyramde (ctg, biotin-tyramide), homnovanillic acd, or 4-hydroxyphenyl cetic acid), generates an amplified signiaL Methods of using GO and HRP in a proximity assay are descihed in, eg. Langry a M U.SA ept of EnergyReportNo, UCRLID-36797 (1999h 2 X he biotin-iyramride iusdathsendmemer oth gnapiication pair, the F1RPH2O 2 complex oxidi zes the tyramide io generate a reactive tyramide radical that covalentiy binds nearby nucleophilic reias The activated ryramidre is either directly detected or detected upon the addition of a signa-detecting reagent such as, for example, a streptavidiniabeled fluorophore or a combination of a streptavidiniabeled peroxidase and a :30 chromogenic reagent, Examples of fluoroph ores suitable for use in the present invention include, but are not limited to, an A.iexa Phuor*' dye (e g Alexa Plnor*555, fluorecein, uorescein isoatocyanate (FlTC), Oregon CreenY. thodamine, Texas red, tetrarhodamine isothioeyaae (TRJTC), a CyDyiNi finor (e.g, CyC3,CS and the like. The $5 streptavidia label cn be coupled direct or indirectly to the iuorophore or peroxidase using ehaods imwnithe avNon4'oning enmples of chromogeni reants sahde fTh tse inthe presentinventon inctende3'5 SwmrnetbylncadaineW (B1 3% dianobenadiae (DAB) 2 no be ethyfbenzothitinessulfbis acid) (ABTS1 4 c hloro- *napthioi (4CN), and/or porphytnogtia jO286]J In another example of proximity channeing, the facilitating moiety is a phontier and the first mmber cf the signal OnpliTicaon pair is a large INole labeled with multiple baptens that are protected with protecting groups that prevent binding of he haptens to a specific binding parmei (e.g ligand, antibody, etc,), For example, the 10 signl 'mpitfication pair member can be a dextran molecule labeled with protected biotin, couiarin, and/or fluorescent mnoleuies, Sitable protecting groups include. but are nti limited to, phenoxy~, analino-, olefin-. thictber-. and sedenoether-protecting groups, Additional photosenstrs and protected happen molecules suitable for use in the proximity assays of the present invention are descid in US Patent No., 7,675. When the 15 potosensitizer is excited with light, it generates an oxidizing agent (A e singtet oxygen) if the hapten molecules are within ebanneing proximity to the phtosermitizer, the singlet oxygenwgenerated by the photosensitizer is ehanne ted to arnd reacts with ihioethers on die protecting groups of the haptens to yield ear boniyi groups (ketones or aldehydes) and sudphicl acid, releasiNg the protecting groups rom the happens. The unprotected haptens are 20 then available to specifically bind to the second member of the signa amplification pair (e.g. a specific binding panrt that can generate a detectable signa), For example, when the haptan is bitin, the specific binding partner can be an enzymielabeled streptavidin, Exemplary enzymes include ailaimne phosphatase, 6-galactosidase, F RP em After washing to remove unbound reagents, the detettbelc genered by adding a detectable 25 (eyg; fluorescent, cherniinescent, cobromogenic,, ecC) substrate of the enzyme and detected using suitable msethods and instrumentation known in the at Altematively, the detectable signal can beo ampAlifiedl uing tyramide sinal amificadon and the' aldvated tyraide eijthea directly detected or detected upon the addition of a signal-deecting reagent as described above s0 W287J in yet another 'ampieofrointyixhannelinggthe factitatinigmoiety is a phosentsiizer and .e first memberof the signal ampiiadion pair isan enzyme-inhibitor conplex. The enzyme and inhibitor (e g. phosphonic acid-labeled detrani) are linked together by a eleavableelinker (eg. gihioetbe4 When dhe photoseneitzers m led wth light n cw6 it generates an xidizng agnt (e., singlet oxygen), If the enzyme-nhibitor comopkIx is within chaumehlng proxrinmiity to the photosensitizer, the singlet oxyn generated by the photosesitizer is channeled ro and reacts with the dleavablet linker, releasing the inalitov front the enzyme, thereby acivating the enzyme. An enyme substrate is added to generate a. detectable signal or alternatively, an amtplificationa reagent is added to getrate art amoptited In a further example of proximity channeling, the facilitating moiety is HRP, the first member of te signal amplification pair is a protected happen or an ezymeW-bbiitor complex as described above, and the protecting groups comrprise p-aioxry phenoL The 0 addition ofphenyenedianine and H2OC generates a r ive phenylene duimine which cnnes to the protected happen or the enzyme-inhibitor complex and reacts with p-aloxy phenol protecng groups to yield exposed haptens or a reactive enzyme, The amplified sin~al is generated and detected'as described above (seseg, U A Patent Nos. -432,13 and 5A,5 944). A 28j An exempiay t pg the prOxit)y assays described herein is provided in Example 4 of PT PubatonV 9 the disclsure ofwh t. hern incorporated by efrene i its entirety otr all purposesN (O29U] in another embodiment, the present tinvention provides kits forn performing the proxinty assays described above comprising: (a) a dlin seres of one or a plurality of 2 capture antibodies restrained on a soird support and (b) one or a plurality of deteetion antibodies (g. a combination of activation state-indpnkrden antibodies and activation state dependent antibodies for detecting activation levels and/or a combination of frst and second activaori s pttendependcm antibodies for detecting expression level in some instances, the kits can funher contain itnstrions for methods of using the kit to detect the expe5ssin 25 and/or activation status of one or a plurality of signal transduction mrieules of cells such as tumor ells, The kits may also conntain~ any of the additional reagents describedi above with respect to performing the specific methods of the present ivention such as, for extuple, first and second members of the signal anplification pair, tyrmnide signal amplification reagets, substrates for the facilitating moiety, wash buffers, etc 30 WVR Production of Antibodies P0291 he generaton and selecton of antbodies not ahead> commerciay avaiable far anayzin the expression and/or activation level ofsgaa transduction moleculesW (ep $17, HlER2 sigating padway components) in cells such as tuonr cells in accordance with the present invention can be accomplished. several ways. For e xample, one way is to express and/or pudfy a polypepide of interest (iantigen) using protein e>:gression and purification methods known toe art, whie another way is to synthesize the polypeptide of interest usng 5 solid phase peptid syncathesis methods known in the mrt See, eag, Guide to Protein Purfwron, Murray P, Deurtcher, . Meth Enzymol, Vi, 182 (1990); Soi d Phase Peptie S e Gr*~Weg A Fields, Aed Math Em~ymot> Vol. 289 (1997); KiOo et a, Chem Pharm BuP &lt 38: 2 99 (1990); Mostafavi er at. Bivoed ept Proteins Nucleit Aetds, 1:255-60, (199); an ujiwara et al. Cher Phanm BIL. 44:13263. (.1996). The purified or 0 symhesized polypeptide cn then be ejected, for exanpe, into rnie or rabbits, to generate polyclonal or monoclono! antibodies. One skilled in the art will recognize that many procedures ae: available for the production of antibodica for example, as described in Antbdies. A Laboratory AMtdual, itaslow and lAne, Eds, Cold Spr ig Hlarbor Laboratory, Cold Spinog Harbor, ThY. (1988), One skiled in the art will also appreciate that binding 15 fragments or Fab fragments which manic (eg. retain the functional biriding region ot anibodies can also be prepared Dom genet information by various procedures See a a Aniibndy Engineering: A Practical Approach, Borrebaeck, lEd, Oxford Un.iversity Prhess, Oxord (1995); and thse at at, j..hn nol, 149:3914-3920 (1992 102921i addition, numrous publications have reported the use of phage display 20 technology to produce and screen tibraries of polypeptides for binding to a selected target antigen (pe, a g, Cwirta a a , Proc, NaL A cad. Sci. 8T6378-6382 (1990); Devon at at. Science, 249:404406 (100); Scott et al Science 249:386-388 (1990); and Ladner et a' U.S, Patent No. 557i698) A basic concept of phage display methods is the establishment of a ph ysicad association bween a polypepide encoded bay the phage DNA and a target 25 ant This p i asocon iS provided by the p1ag particle, which displays a polypepade as pat of' a cpsid encosig the phage geme wIch encodes the polypeptide. The establishnt of a physical association beween polypepties and their genetic material allows sinutaneous mass screening of very large numbers of phage bearing different polypeptides& Phrage displaying a polypeptide with affinity to a target antigen hind to the 30 target antigen and these phage are enriched by afnity screening to the target antigen. The idenity of poiypeptides displayed trm these phage can be determined from their respective genomes. Using these methods, a polypeptide identified as having a binding affinity fox a 8 cesied target atigen can then be syntherdzed in fatdk by cretona1leanse ag U Patent N! 6,05109,4 [02931 The antibodies that are nerated by these methods can then be selected by first screening for affinity and specificity with the purified polypepide antigen of interest ant, if requireT nompaing the resuNts to the affinity and specifcity of the Anidies with other peptide antigens that are desired to be excludedrrom binding Th screening prochure can invov immobiization of the purified poypeptide antigens in. separate wlls of mireoe tr phates, The solution containing a potential y up of antodies is then placed tito the respective microtites wels and incubated for about 301 moimaes to 2 houtv. 10 The mieatter wells are then washed and. a labeled secondary antibody (",gt an antimouse antibody conjugated to alkaline phosphatase if the raised atubodies ae moose anibodies) is added to the wells and incubated for about 30 minutes and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immirobilized polypeptide atitgern is present V t{1294f The antibodiesso idenuiedcan then be further anayzed for affinity and specrhiity. In the development of immnuacassays for a target protein, the puritied target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the anibodOie that have been selected, Because the binding AnY OV' various antbodies may differ, certain antibody combinations may interfere with one another sterically, assay 20 pcrformanc of an antibody may be a more important measure than absolute atfmity and pec toy of tha Iantibod {02951 Those ski lad in the art will recognize that many approaches can be ken.i producing antibodies or binding fragmens and screening and selecting for afi and specific for the various poiypept&s ofinteret btthese approaches o not cange te 25 s present ntViTr A NPeycIota4 Antbtdlias (02961 Polyd onal ant:hedis are preferably raised in animals by nxulti ple subcutaneous (so) or intraperntoneal (ip) injections of a polypeptide of interest and an adjuvwant, It may be useful to conjugate the polypeptide of interest to a protein carrier that is immunogentc in the species 30 to be immumzed, such as eg, keyhole limpet hemocyanin, seum albumin, bovine thyroglobuhst or soy bean trypsin inhibitor using a bifuntonal or derivatizing agent Non limiting examples of bifuncionm or derivatizing agents include .mdeimidobenzoyl uvffmccinktnde ester coatien though eysteuie reidues).Nyatysuccitmide (eonjugation through lysinerenueutaadehyde succiniecinhydride~ SOd e.and RN= N. wherein 1andt are dirffenrtaiky groups. 102971 Animais are imnmUnized against the poy pepide of interest or anfiunogen c conjugate or derivative thereofby comhining. & . £s00 pg (for ra bbits) or 5 psg (for mnice) of the antigen or corugate with 3 volumes of Freunds complete adjtvant and injecting the soluttin tntradermady at multiple sites. One month later, the animan am boosted with about 1/5. to 1/10 the original amount ofpolypepide or conjugate in Feretd's Wcomplet vant by subcutaneous injection aY multiple sites. Seven to fourteen days later, the animals are bied It and the serumn is assayed for arnibody titer, Animals are typically boosted. until the titer lateus. Preferably, the animaE is boosted with the conjugate of the same polypeptide, bat Co jugation to a dieet inogerdc protein and/or through a diaernt ossdinng reagern may be used. Conjngates can also he madec in recombinant cell culture as fusion. pltotetas. In eerta in taces, aggreg atig ants such as ahn can be used to enhance te f%. Mutnoecina Antibodies [0429833 Mooconal antibodies are generally obtained from a population of sstatially homogeneous antibodies, ia the individual antibodies comprising the population are identical except for possible nattnailyoccurring mutations that may be present in minor 20 amont Thus, the modiner "monotlona A" indicates the character ofthe antibody as not being a mixture of discrete amibodies. For example, monoclonal antibodies can be made using the hybridoma method described by Kohier at dotuNre, 256:495 (1975) or by any recombinant DNA method known in the art (sc, e.g. U.' Patent No. 4316,567). [i299 in the bybridoma mrhod, a mouse ot ther appropriate host anuima (e.g, hamster) 25 is in10unized as described above to elicit lymphocytes that produce or are capable of producing antibodies which spec ifically bind to the polypep tide of interest used for immnuiation, Ahtemativey, ynmphoctes are .immnunized in vitra, The immnunized lymphocytes are thea. fused with myra cell~ Is using a suite fusing agent, such as polyetbyiene glycol, to form hybridoma cells (see, e.g, Goding, Mlanodonwamatbodies: 30 Principles cad Practice, Academice Press pp, 59- 103 (1 986)) The hybiddoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substancec which inhibit the growth ur survival of the unhised& parental roy om cells. 990 for exampKedtM parentabl w oema calls lack the cnzyma.ehypoxanthine gimin phlospthodbosyl tr anese (HOGPRT). he colite mued lum for the hbrdorma celskv1il typicacy inchaiypoxnthbe aminopten dad 1lsndine (HAY' medium) which prgeen dhe cmvth of HGPRTidcicient cels 5 L43001 Preferred myeomna cellS are those that fAtse efficetly, support stable nthigh-lvet poduction of antibody by the selected antibodyproducing cells, a/or are selitive to a mecdiumn such as HAT mnedium,. Examples of' such preferred mxyeoma cell lines f or the productions of hman monoclonal antibodies include, but are not limited to. marine myeloma lines such as those derived hum MOPCM2 and MP& I1 mouse turmors (available frm the 10 Salk Istitute Cell Distibution Center; Saniego, CA), SP- or X63-Ag653 cells (available from the American Type Culture Collection; Rockville, MD), and human myeloma or muse-haman heteromyeloma ceil lines (see, a. g, Kozbor, Ii !smun.. 133:3001 (1 984); sad Breux et at. Mdhoceda Anatibody Proadaton 7Techniqfues and A applications, Marcel Dekker, Inc. New York, pp, 51-63 (1987)I S-153)t The culue medium in which hybridoma cells are growing can he assayed I the production omnu lunal antibodties directed agai th polypeptide of interest. Preferably the binding speificity of monokonal antibodies produced by hybridoma cells is determined by p tti . or by an in via binding asy, such a a radioimmuoasay (RdA) or an r yme-e~iiked inrnooabsorbent assy (EllhS.A), The bindingp atlinity of monoclonal 20 antibodies can be determined using, e g, the Scatcnard analysis of Munson at at, AnaL Bochen ,Ih? (19$ (0302) After hybridomra cells are identified that produce antibodies of the desired specificity, affiity, and/or activity, the clones may be subeloned by limiting dilation procedures and grown by standard methods (see, extg, Geding. MonconaldAntbodies: 2$ Principhes and ?mectice, Academtic Press, pp, 59-4(03 (1986)),~ Suitable culture media far this purpose include, for enx mple. D~M.M or RPNI-1 640 medium. In addition, the hybridoma cells may be grown in vtro as ascites tumors in an arnmal, The msonoclonal antibodies secreted by the subelones can be separated fromi the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, 30 hydroxyiapaite clhromsatography, gel electrophoresis, dialysis, or affiity chromatography, 1t3O31 DN encoding the nonodounal antibodies can be readily isolated and sequenced using coinvention.al procedures (egby using cligonuclectide probes that are caeale of 91 binding specificaly to genes encoding the heavy and liglt chains of urine antibodies The hybridoma cells serve as a preferred source of3 such DNA, Once isolated, the DN A may be placed ino xpression vectors, which are then transfected into host cells such as .2 colt ceLls, simian COS eeis, Chinese lamster Ovary (CO) cells, or myeloma cells that do not ) otherwise produce anibody to induce the synthesis of monoconal antibodies in the recombinant host cels, She es. Sierra e at, Curr. Opin.Thmmunat, 5:25&262 (1993); and Pwuckthurr, bhnunoi Rev.. 130:151-188 (1992). The DNA can also he xmodnfied for example. by btutin the coding sequence for humna heavy chain and light chain constam domains in place of the homoldogos mnurine equences (see, e, lUs. Patent No, 4,8 16,567; and 10 Morrson et al, Proc.?Nat. Acad Si TSA 8:6851 (1 984)), or by covakntly joining to the in~ujnolobulin coding sequence all or pat of the coding sequence for a non immaogflobouhl polyeppide. [03Q4) In a further embodiment. monceonral antibodies or antibody fmgmemans can he isolated fromR antibody phge libraries gererated using the techniques described in for 15 example, McCafferty 53:ature,3485-5.4 (199) Ciackscn at al Ahare. 352:624 628 (199); and Mark:stal, M Bitt, 222:5&1-597 (1991) The production of high affinity (nM rango) human monoclonal aMibodies b eban huffing is described in Maks e at. .iboTechnology, 10:77983 (1992) The use of combinatorial infection and in vira recomination as a strategy for constructing very large phage libraries is described in 20 Waterhrouse et a, sc, Acids Ra, 21:S-25266 993. Thus, these techniques are viable alternatives to traditional monclnal anybody hyhdoma methods for the generation of noCnoionad antQWii C( Humianized Antibrdies {@3051 Methods for hun anizing non-human antibcdes arc kuown in the art. Preferabiy a 2.5 bmnanized antibody has one or more amino acid residues introduced into it from a sour which is non-htana. These non-hunan saminn acid residues are often referred to as "Nmport residues, whihb are typically taken fronn a import" variable domain. Phumanization camn be essentially perfOnned by substituting the hypervariable region sequences of a non-human antibody for the eorsponding sequnces of a human antibody, Se, e.g_ Jones e at 30 Nanue-. 321:522-25 (1986); Riechanaun at at. Natre, 332,323-327 (1988); and Vethocyen t at Science, 239:1534-4536 (1988). Accordingly, such "humanized' antibodies are chimerib antibodies (see, eg U.S. Paent No. 4.816,5671 wherein substantially less than an 9Z2 ntact hmman varIabe domain has been subhstitmed by the gore spondina sesnentrn noiniuan species.I ractic h rumanad ntibodiesae typicanyyumin exrdhbdies n wthI Somehyp raraab rgioa redes and possby sonic frauewok regi FR) resdtes are substituted by residtres fom analdogoishsies ofdrodont atibodies. S 16306) The choke of human variable domains, both light and heavy, to be in making the bmanized antibodies described herein is an. important consideration fr reducing antiaenicity, A cording to Qh so-called " method, t sequce o variable donan of a rodent antibody is screened against the enire library of known hum an variable domain sequrece, The huan sequence which is closest to that of thbe rodent is then 10 accepted as thehuma FR for the humanized antibody (see, ag, Sims et at, I .bnmunrrt, 11 i:2296 (1993); and Chothia et at, £ Mol. Biol, 196:90 1 (1987)) Another method uses a particular fR derived from the consensus sequence of all human anibodies of a particular subgroup of iht or heavy chaims. The same FR may be used for several different hurnanizedi antibodies (seae e. Carter et alt, Proc. N att scd U ,94285 (199.2.', and Jresta e 5 ,.1 hm unoat 151:2623 (1993)). [03071 i is also important that antibodies be humanized with retention of bigh affiity i the antigen and other forableioogica properties, To achieve this geal, humanized antibodies can he prepared bya press of analysis of the parental sequences and various tonceptnal humanized prodnts using thredimensional models ofthe parental and 20 humanized sequences, Three-Aimensional immnoglobulin models are emnmonly available and are famuiar to those skilled in the art, Conmpuer programs are available which illustate and display probable three-imrensional conformnational structures of selected candidate immutniglobuisi sequences, inospectio~n of these displays permits analysis of the likely role of the residues in the functioning of the candidate iammuoglobulin sequence, Lae. theanyiso 25 residues that infueme the abiiitv of the candidate imunogiobuhn to hind its antigen In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic\ such as increased affinity for the target antiets is achieved Inla eeral, the liypervariable region residues are diretly arnd specifically involved in influeina antigen binding. 30 j03(8j Varsons oros of humanized antibodies are contemplated in accordance with the present invenion, For example, the humranied antibdy can be an antibody fragment such '*> ad '-v can, be, an intact antilo- m as ab fragment. Ahativelithe .mmanzed anhd e e nhe niodyxsuch s an intact4 ig(3g, or 1gM antibody. D Hmmnai Aathodis [03&91 As an ahernative to humanzation, human antibodies can be generated. In some 5 embodimens, transgexic animals (eg. mice) can be produced that are capabe, upon imtiati, of producing a full repertoire of hinnan antibodies in the absence of endogenouns inunsunoglobulin production For exanmpie, it has been described that the homozygous deletion of the antibody heavy-chainjoinirg region (01) gem in hineric and ger m tine mutant mice resultss in copeeihbto fedgeosatbd rdcin 10 Transter of the human gemiine immunoglobulin gene ay in such gem-line mutant mice will result in the production ofthuman antibodies upon antigen challenge, Se,. eg., Jakobovits e: al e Prac Nat Acad Sc. USA, 90:2551 (1993); Jakohovits er alt Nange, 362:2525 (1993); Brugermann at at. Year i n immn 7:33 (1993); and U.S. Patent Nobs SA9669, 5,89,369, ad 5,545, 1.5 03 ) Alteratively phage display technology (see, eg McCatferty er at, Mu re, 348:532~33 M99)) can on useJ to produce human antibodies and antibody fragmems in rXYl donga vitr, using im muniogzobulin variable (Vroain gene repertoires from tnirmmuinized donors, According to this techiupe, antibody V domain genes are cloned in-fmmne into either a major or minor coat protein gene of a .ihunentous bacteriophage, such as vi 3 Or fd 20 and displayed as functional antibody tbrgments on the surface of thre phage particle, Because thet tibunentous particle contains a single-strandedi DNA copy of the phaege genome, selections based on thbe firnctiona properties of the antibody also result in selection of' the gene encoding the antibody exhibiting those propertei. Thus the phage mirics some of1 the properties of the 13 cel. Phage display can oe perfmned in a variety of formats as desrbed 2$ in, engi Johnson et a!, CurO4tn. Struct .Biot 3:564,571 (1993h Several sources of V gene segmrents can be used for phage display, Sere eg. Clackson et alt Natwerr, 3.52:624-628 (1991), A repertoire of V genes fo unnmounied human donors can be constructed and antibodies to a diverse array of antigens (incinding self-antigens) can be isolated essentially following the techniques described in Marks ep aL,1 Mo1 Biot, 222:581597 (1991); 30 Grifith e a., EMBO 12:725-734 (199; and US. patent N 5os. 5565.32 and OW7 , 031 1 In certain itae human antiodes can be generatedb o activated B cells as described' in g US;Patent Nos. 5,56 9610 and 54229575 F5,. A~afibody Fragmnts N11 Various tehaques have been developed for the production of antibody fraent, et Jtac, a a e o Ci Traitiomiythese fragorens wexre derived via proteoltic diti<o of inac atibodies (se e'' Mortom et a J Biochem Biophys. Atk 24:107-i 17 (19921 a d Btoan er d t S&ience 22: 81 (1985)), However, these fragmnets can. now 'be prouced directly using tecmbtean a ot clls. For exnzlple, the anybody fants can be isolated from the antibody phage libraries discussed abov'e. Alternatively, Fab'SB Ifragtets can be directly recovered from. J Fol cells and chemically coupled t foron F(ab t ' lagmntc (see, e>g. Carter et a, BDio.Thknology, 10:463-167 (1992)), Acc.ording to, another aipproach, Ptabh 10 fagment ca be isolated directly om reombin host cel culture, Other trechnique for the producton antibody fagments wo be apparent t hose skIed in the art in ohe. embdi meta, the anti y of cc is a singklehn Fv fragm (cv See egPCT Publiumion No. WO 93/161g5; and U. Pateot Nos '571 94 and ,45, lT ,a9ody ftrament may also be a linear antibody as describe. e.. in US. Patent No. 5,641.870. Such 1 linear antibody fragments ay be monospecitic or bispecic F. Bispeile Antibodiec [3t3] ispei3 c atib odies are antibodies that have wn speitficis for at ,east two ddfebrent epitopes. Exempnlary bispe'cific ac'tibodies may bind to two different epitos of the same polypeptidec of' interest. Other biseelf tib~odies may comrbine a binding; site for the 20 polypeptide of interest with binding si for one or more additional aptigens. Bispec e antiod'es can be prepared a fulidenth antibdies or anti body frat (&g, F~ab' bipecifie antib 'dies), [83c~141 M~ etds frmaing bispecific antibodies are know inh the art. Traditional production dof ful egth bis'pecifle antibodies is based on the o-expression of two 25 imunwogobuin heavy chain-light chain pairs where he two chains have different xpCitites (see, e Mlsteit aL, Nate, 30:537-539 (1983)) Because of the andom assortment of mimnoglsobudin heavy and Ughltchains these hybridoma (quadromas) produce a potnal mixture of 10 diflorent antibody molcule2, of which only one has the connect bispecifi stetra e PuArification of te uone nolecue is usually performed by 30afllnity chromnatagraphy. Simihur procedures are disclosed in PCTI Publication No, WCI 93/0882.9 and 'Thrunacke. r t. ZEBOJ 10:3655-3659 (1991).

P33151 According to a diffrernt approach, antibodyvaria ble drains with the iesintd binding specificities (antibody-antigen cmingsites) are iused) to imngoui conlstamE domain sequece, ITh fon preferaly is with an immnlobuinheavy wbain constant domain, conprising at least part of the hinge, CH2, and CH3 regions, it is preferred 5 to have the first heavy chain conant region (CI 1I containing the site necessary for light chain binding present in at least one of the fusions, DNA encoding the in uuoglobulin heavy chaix fusions andI, if desired, the immn tglobulin ligt chain, are inserted into separate expression vectors, and are c ansfected ino a sMabe st organism. This provides for great 1eli ty i adjusting the mutual proportions of the three polypeptide fragments in u embodiments when unequal ratios of the three polypeptide chains used in the constmtion prove th optimum yields. t is, however, possible to insert the, coding seqeces fr tvo or all three polypeptide chains into one expression vector when the expression of at hes two polypepide chains in. equal res results in high yields or when the ratios ea of no particular 15 {03t In a preferred embodiment of this approach, the bspecific antibodies are composed of a hybrid irnAunogAoulin heavy chain with a fist binding specificity in one arm, and a hybrid immumnogiobnlin heavy chaimlight chain pair (providing a second binding speciticity) in tthe other aarn, This asymmnetric siructure facilitates the separation of the desired. specific compound from unwanted immunoglobulin chain combinations as the presence of an 211 irnrmunoglobuiin light chain in only one half of the bisecfi mlecule provides for a faceile way of separation Sea, ae, PCT Publication No. WIO 94/04690 and Satesh at al. Melk .Emnofa 12) 210) (1986). 103171 Aocozding to another approach describe i US Patent No. 5,731,)68, the inerface between pair u<' antibody mnolecuies can be engineered to mainmize the percentage of 25 beterodirners which are recovered orm recominant cell euure, The preferred interface comprises at least a part cof the CH3t domsain, of an antibody constant domain. in this method, sne or more amal) amine acid side-chains frm the imterfae of the first antibody molecule are r with larger side chains (ag tyrosine or iryptophant Compensatory "cavities" of identical or similar size to the large side-chainPs) are created on the interface of the second 30 antibody molecuc< by replacing large amino acid side-chains with snaiet ones (e.g. alanine or threOnine). This provides a mechanism Ar increasirag the yield nf the betemdimer over other unwanted end-prducts such as homodimers, [03181 Btspecatz antiodies incide crsslinked or htcontuate" antibodies. ar exanipm w one of the anCies n he hetarconjngata can be coupled t avidin the other to bictn Hteterocanjugate antibodies can be inade using any convenient osseinkirng methaa. huitahia crosslinking agents and techniques arevidaknown in the art and ae disclosed in 5 aU. atentN 4,76 , [0319j Suitabic techniques for generting bispeeltic nibodies fRom antibody fragments sa also known in the at For example, bispecific antibodies can he prepared Msing chemical linkage, In certain instances, bisp elc antibodies can be generated by a pocedure in which intact antibodies are proteolytically cleaved to generate F(abh fragments (see, e,z Brennan t0 etd Sce4 2291 (19T5)), These fragmnts are reduced in he presence of dhe dithiol complexing agent saditne arsenite to stabilze vicina dithiots and prevent interi uar disudtiea formatio. The Fah figments generated ar then convened to thionitrobentzoate (lTN) d4e;Nrv e One of the FbThTNB derivatives is ten reconverted to fhe Fab'thia hy reduction with mey ae and isnixed with an. equmlar amount ofdte other $ Fab~ TNB3 derivative to form the bispeciic antibody, [032tI in some b odmnFts. Fob SH fragments can be directly recovered fro EJ cl and chemnicaly coupled to fkmn bispecific amibodies, For example, a fully humanized bispecitc antibody Frabt molecule can be podneed by the methods deserbed in Shalaby a <al j Exp. A'd, 7 - 2 17-225 (1 992) Each Fab' fagmet was separately secreted Iomn I 20 eoli and subjected to directed chemical coupling in r to Sorm the bispecific antibody 0321 iou echiuesifr maing and isolating bispecifie antibody fragmeas dctdy frm recomibinant cell culture have also beam described, For example, bispsecific auntodies have bean produced using leucine zippers. See, ag, Kosteiny et d.. !mmucno, 148:1547 1$$3 (1992). The eucine aipper peptides from the Fos and Jun poteinsvereinked to the Fa> potions of two different antibodies by gene fusion, The antibody homodimers were reduced at the hinge region to form monomers and. than re-xidized to torm the antibody haterodimers. This method can also be utilized for the production ofantibodv homodimets. The "diabody"~ technology described by Hollinger er dt Proc. Nat, 4cad Sci. USA, 90:44n44 (193 has provided. an alternative mnechanismn for making bispecific antibody 30 fragmens. The fragments comprise a heavy ehein variable domain (VH) connected to a iAh chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on thre same chain, Accordingly, the VH and VL domains of one fragent are 97 frcrced to rair with the comuplaeemasyVL and V14 domains of another frngthentthertb rrming two antigen binding sies Arother raiogy for maki g bispecifi antibody wagment by the tse of single-cainf Es) dracrs is descrbed in CGrber tL hara M e:5368 (t9941i 5 {f622] Antibodilesith more than two valcncic C'fre also cottniteld< For exatnpic Atrispecic antibodis can beepared se a Tit a t , 8101M, 1: 46- (199 . C, Atbody Purituthtn [0323{ When using recombinant techniques. aibdiescan be produced inside an isolated host cellk in the petriplasmic space of a. host cell, or directly secreted from a host cell into the 10 medium. if the antibodyv is produced imtraceluiatdy; the patrticulate debris is first removed, for exampke by entrifeigation or ul taitration. Carter et al. RWiTeelc& 10:163-167 (1992) describes a procedure for isolating antibodies which are secreted into the periplasmxic space of It collt Briefly, cell paste is flawed in the presence of sodium acetate (pH '45, EDTA, phenyhnsethyhnltonylliuodrd (PMS F) for aboutt30 mrin, Cell debris can be removed by $ centrifation, Where the atntibody is secreted into the medium, supernatants from. such expression ssems are genemaly cancentrated using a commerasy aable protein concentratiott fiter, tot example, an hAmion or Millipore Pellicon ultratiltration ni A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteoysis and antibiotics mays be indhided to prevent the growth of adventtio us 20 contaminants. O324) The antibody composition~ prepared from ceilts can be purified usIng, for example, hydroxylapatite chromatography. gel electrophoresis, dialsiss and affinity chromtography, The suibility of protein A as an affinity ligand depends on the species and isotype of any imanoglobuhn Fe domain that is present in the antibody. Promn A can be used to purift 25 antibodies that are based on h umnan l., 2 or y4 heavy chains (eer eg. Landmark er a?., 62:1-13 (1983)) Protein C is recommended for alt mouse isotypes and for human 73 (see g,.uss el at. EABCi 5:1567-1575 (1986)), The matrix to which the afiniwtgand. is attached is most oten agurose. but other matices are available. Mechanically stable matrices such as controlled pore glass or poiy(styrenedivinyl)benzene 30i allow for faster flow raes a srter processing times tron can be achieved with agarose, Where the antibody comprises a CH3 domain, the Blakerhond ABX resin (J" T. Baker; PhillipbUrg, R,) is usefdu fir purification. Other techniques for protein purication such as 98 nractnation On . exchange caann, oianl preipltation revert phase RW.UX chroattapy edsiica, dumatography on hepain SEPHtAROSEngX totun agraphy on ananon oaton exchange resin such as a polyaspanidc acid cMonst, tomatotocsing, SRAGEK ad annmomni sulfate precitation are o available pending onthbe anybody to be covered. [9325 owi an niiay publcation step~) )he mixtuevransng the antibody of interest and conianinaats may be sobee to how pH hydrophobiceinteraction chroay using an celidon buffer at a pH4 between about 2.44 preferny performed atk salt ticentiations (g fiorn about 0- 31 ab. 0 [0326) One of skill in the art will appreciate that any binding molecule having a fbnction similar to a antibody, e&g a binding molecule or binding partner which is specific for one or more anaytes or interest an a samplne, can aliso be used in the methods and compsin osof the present invention. Exam!.ples of~ suinbl antibOdylike mnolecules inle bu are not litedxtcc to. dtomino antibodies unibodies, nanobodles, shark antigen reactive proteins, 15 avies, adnectsins, antica'ms, affiiy ligands, phylamners aptamners, antibodies. trinrcdns anIhe like. IXK Mthods of Admiulstation 10327] Aneording to the th~tods of the inventi the HELR2-modultn copounds and other anticancer drugs described hemin (colcutiveiy "anticaucer drugsf are administered m a 20 subject by any convenient means known in the ar. The methods of the present invention can be used to determine or predict the sensitivity of a cell (e~g, a tumor cell) to treatment with an. anticancer drug such. as a H ER2-modulating conound or a combination of anacer dgs, The methods of the invenion can also be used to determine, predict idetif andor mnitor the response of a tumor (g, a breast tumor) to treatment wdth an anticancer dr 25 such as a HER2-modulating compound or a comnn ation of anticancer drugs. The muethos of the present inventi can fariter be used to select a suits ble anticancer drug such as a E2,tMduaring compound or a combination of anticancer drugs for the treatment of a tumor (e.t tumor) ina subject. One skilled in the art wil appreciate that the aniactdrugs described herein can be administered alone or as part of a comnbined 3 therapeutic approach wit conventional chemotherapy, radiotherapy, homanal therapy; immnunotherapy; and/or surgery, 99 (03281 I certain emtbodimrents, the antleancer drsug comprises an antistgnalin agemt (ha a cytostatc drag) such as a rO a antibody or a tyrosine kinase ihitor;ananti proliferative agent; a chemothwnrpeutic agen. (ia., a ytotPoc drugb: a hormone therapeutic agen; a radtatherapeuti agent; a vaccine; and/ anany other coMound with the ability to reduce or arogate the uncontrolled growth of aberrant cells such as cancerous cells. in somec emibodiments, the subjet is treated with one or more anti-signaling agents, anti-prolifrative agents, and/or hrnonal therapeutic agents in combinaton with at least one chemotherapeuti agent, Examplary mnocional antibodies, tyrosine kinae inhibitors, anti-poiftetive agents, chemnodhrpautic agents, hrona therpticaents radotherapeutic agents, and vaccines I are desetibed above [32 n n particular embodiments, the anticancer drug comprises one or more compounds that modulate HEBR2 activity including monoclonal antibodies. tyrosmne kinrse inhibitons. and combinations thereof Nondtimiting examnples oflHER2-mnodulating comrpouinds include maonoeonal antibodies such as rastuzuman (Hercepti*) and pertuumah (C4); small 25 molecule tyrosine kinase inhibitors such as geftinib (Iresra) erltinib (Tarcev"). pilitinih CP~5457,C.P-724714, canertinib (C1 1033), H1KV272X apatni.b (GW-5206; Tykerbs), PKI-66, AtE88 BMvS-599626, IAXI3 57, BIBW 2992,M AY345\43 J tN]26483 327, andi JNJ~2648'3 327; and combinations thereof. n certain embodimnts, HER2modulting compounds can be used in combination wit one ormore other anticancer dugs described :0 herein or known to one of skill in the artt {033I1 In some emboditnuts, the anticzancer dmgrs described herein can he co-administered with conventional immnunotherapeutiw agents including, hut not limited to, immunostailants (atgz Bacillus Calmette-Ciusrin (BO) I levamisole, interieukim-2, alpha-interferont ete) iunmmotoxins (ag -. anti-CDi33 nmonockmal. antibody-calicheamicin con jugate, anmiCD2:2 25 monoclonal antibody-pseudomonas exotoxia conjuagate, ae,) and raioimmumnotherapy (eg, anti-CD20)r mnocional antilody conjugated to 'In, y, or etc i0331i Anticancer drugs can btecldnistemed with a suitable phanrnaceutical excipiet as necessary and car be carried out via any of the accepted modes of administration, Thus, administration can be for example, oal buccal, sublingual, gin givL pahal intravenous, 30 topical. subcutaneous, transcutaecous, tranadermal, intrarmuscular, inftalot. parenteral, intra-astealrde, intradenxnai, inraventricular, intracranial, untraperitonealk intravesical, intahecal, intraiesiona,. intranasaL rectal. vaginal or by inhalation. By "co-admainister" it is meant that an andicancer dr is adminisered at the samne timre just prior to, or jut after the adtmnistratint of a second drug (e. another anticancer drug, a drug useful tor reducing the sieeffcts associated with anticancer drug therapy, a radiotherapeutic agem, a hormronal thersautic agent, an immu wnothrapetuci agent, etc). 5 [0332] A thetapeuticll e~eetive amort f an anticancer dirug mays be admninstered repeatedly, org at least 2, 3. 4, 5, 6, 7, 8, or more times, or the dose masy be administered by continuos intbcsion, The dose meay take thre fr of' solid, semei-solid, lyophilised powder, or liquid dosage forms. such as, for example, tablets, pills, pellets, capsuks powders solutions, sS creams, oimments. lotions, es, aerosols, oams, or the like, preferably in unit dosage forms suitable for simpe administration of precise dosages. 1033-3 As used herein the term 'mit dosage form" refer to physiraiy discreIte units suitable as ,ntr doas for humani subjects and other mammnals, each unr contaiing a predetermined quantity of an anticancer dmrg calculated to produce the desired onset, 15 tolerability, and/or therapeutic effects, in association with a suitable pharmaceutical xcipient (et. ansrmoule) h * ~ . Inaddition, more concentrated dosage formns may be prepared, from which the more dilute unit dosage forms nay then he produced, The more concentrated dosage forms thus will contain substantially mome than, e.g, at least. 1, 2, 3, 4, 5, 6 , , 9 0 m or more times the amnt of the' anticancer drug' 20 10334 Me thods to.r prepare such dosage forms are known to tiose siDed in the art (see, er Ram mN S I <ca 4 SwasCS, 18t Eri Mack Pubiishing Co., PEton,. PA N"'A 1990)). The dosage forns typically include a conventional ph4oarmcutical cancer or excrepient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhances, soluiizers, and the like. Appropriate recipients ca be tailored 25 to the particular dosage ftorn. and route of administrations hby methods well, known in the art (see. a;., Rrismowhv s PttuRHCwrica Sctzycp>& supta)l. 0335] Examples of suitable excipiens include hrut are not limited to, lactose, dextrose, sucrose, sorbitol, mannitoL starches, gumu naac a alciu phosphate, alginates, tragacanth, geclatin., caleiumn silicate, miicrocrystaline cello tSse, poyvi nylpyrrolidone, cellulose, water, 0 salinenp, methyleeliuloae, ethyleelluloae, hydroxypropylmiethylee&Uulose, and. polyacrylic acids such as Carbopoia, g. Carbopol 94., Carbopol 98. Carbopod 98L etc. The dosage forms. can additionally inceude lubricating agents such as tale, magnesiumn stearate, and 101 ninereloil; weintg agens; eunmigilng agents; sending agents; presrting agents uch as meihvl~.ethyl> and p pyiydrosyben ate~s (e 5 the pambens);p1 adjustng agents such asbotganic and organs acids and base; sweetening agents; and thudng agens the doae farns mays also coniriautobodegradable podymer beads, dsstrans and cyciodextdin 5 insion complexes, [3361 For or4 administration, the therpemically effective dose can be in the form of tablets, capsules, emuins, susnsions, sxion ps, sprays, loenges, powders, and sustainedrelease tbrmulations. Suitable recipients for oral administaionicude pharmaceutical grades ofmannitol, lactose, starcI, magnesium searate, sodium saccharine, 0, talcum, ce ulosq glucose, glatin,. sucrose, magnesium carbonate, and the like [0337 in some embodiments, the therapeutically effective dose takes the form of a pill, tablet or capsule, and thus, t dosage foo can contain, along with an atnticancer drug, aly of the ollowin: a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a disintent such as starch or derivatives thereof; a lubricant such as mnagnesiu stearate and 15 the like; and a binder such a starch, gun acacia, povvnylpyrrolidone, gelatin, cellulose and derivaties thereof An anticancer drug can also be fonudated into a suppository disposed, or ample, in a polyethylene glycol (Pi1 career, [0338 Liquid dosage [orms cla be prepared by dissolving or dispersing an anticancer drug and optional one or more pharmaceutcally acceptable adjuvants in a carrier such as, for 24) eample, aqueous saline " e g 09% witv sodium chloride), aqueous dextrose, glycerrd, ethanol, and the like, to fM a solhtion or suspension, e.g:. for oral, topical, or intravenous administration. An anticancer drug can also be formulated into a retention enema. N0339i For topical administration. the thenapemically effective dose can be in the form of cemuls ions, lotions, gels, toamts, creams, jellies soluions, suspensionls. ointments, and 25 Ransdermal patches For administration by inhalation, an anticancer drg can be delivered as a dry powder or I liquid form via a nebuizer, For parenteral administration, the therapeutically effetive dose can be in the form of e injectable solutions and sterile packaged powders, Preferably, injectable solutions are formulated at a pH of from about 4.5 to about 7.5 03401. The therapeutisiny et ie dose can aso be provided in a Iyophilized form. Such dosage torus may include a huffer e biabonate econstttion pn'or to administration the buffer may beinchuded inthe yophilizeddosage artn for reonstittion wit, e>g water. 'The iyophilized dosage form ray further comprise a suitable tage The lyophized dosage form can be provided in a syrnge uptionaldly packaged in camrbinatiox with theC buffer for t(econstitton such that the reconsituted dosage form can be immuediateWy administered to a subject. 5 0341 A subject can also be monitored at periodic time interals to assess the eficacy of a certain therapeutic regisea. For exFampe, the activatin states of cenain signal transduction molecules may change based on the therapeutic effect of treatcen wit one or momr of the anmicancer drs deserhed heroi. The subject can he monitored to ases response anid understand the effects of certain drugs or treatments in an individuhzed appoach, i) Additionally, subjects who initaly respond to a specific anticancer drag or combination of anticancer drgs ray become refractory to the drg or drg combination indicating that thesc subjects have developed acquired dog"" reisane These subjets can be discoinued on their current therapy and an altenmative treatment prescribed in accordance with tae methods of the present invention. |W342N uncertain aspects, the methods described herein can be used in conjunction with panels of gene expression rarkes that predict the likelhood of breast cancer prognosis andi/toro recurrence in various l of women wh fr e p n ngat di Thesegene anels can be usefl ter ientifying women who are unlikely to exprec 'fcannn beersenceA~n recurrence and, thss unlikely to benefit from adjuvanrt cmotherapy, The expression panels 20 can be used to identify women who can sally avoid adnvam chemtherapy, without negatively affecting diseasetee and overall survival oucomnes. Suiabte systems include, bat are not limited - Oncotyp DX t which is a 2 -gene panel from enomic Health nc Mammnatrinat? which is a 70h-gene panel from Agendiat and a 76-gernc pael from Veridex, [0}343j Ta addition, ini certain other aspects, the methods deanried herein can be used ini 2. conjunction with pariels of gene expression markers that identify the o ignl tumo for cancers ofunknown primryThe gene panels cn be usefid in identifying women with metastatic cancer who would benefit from therapy consistent with that given to wonen diagnosed initially with breast cancer, Suitable systems incinde 5 bm are not himted to. the A-visa CaneerTYPE iD) assay, an RT-PCRbased expression assay that measures 92 ge-es to 30 identify the primary site of origin for 39 tumor types; and the Pathwok Tissue of Origin Test which measures the expression of more than 1600 genes on a icroarray and compares M tumors gene expression "signature" against those of 15 known tissue types " I03 103441 The fHowing exampleaeoiffered to i sta but no to liitthrelaimed [0345 he Examplic n PCT Pulicatio7No<12009/(h37are he;inca nd $ by refence in their entirety for l purpose's EXupie L Deteion, Enameraion, and Characteriaation af Circulating TumAr Ce a (CTCs) in Cancer Patients ith Magnetic Captr and lighSensitivity htmanoassay 103461 This example illustrates a study perored on clinical samples in which the prx1nity assay descibied herein was validated u sing CTCs isolated from cancer patients. 10 [PO47] The inclusion criteria for enrollment in i sudy were as folows (l) older than 98 yeas; (2) histologically conrmeda slid cane (Stage hb or 4); and () tage 3b breast or lng cancer with LN stagingTN, N or N3, IT exu sion criteria fr enrollment in the study were as follows: (1) i 8 years or younger; (2 without mretastasis (3) diagnosis of prostate catncer or melanomoa; (4) prioe history of other cancer within last $ yar; and (6) Stage 3b breast or ing cancer with LN staging f .NX or NO, 103481 20 IN of whole blood was collected by venipI)nure as flows: (1) I0 ml in a CellSave Preservation Tube; and (2) 10 ml in an ED!A Vacutiner Tube The sampLs were shiped by FedEx on day of onliection at ambient temperature, The case report frm rcilded the age gender, ethniciy cancer type current and previous therapies concnAmiant 2D meicm t and adverse events For breast cancer patients. ER, PP, and HER2 satus were akas included. j9] A tofcT2 sujtcts oosspondto 100 canceer types (inciding3 with breast cancer) and 25 eonfrobs weye enrolled in he study. Patient demorgrpiicare provide ia ableale 4 Etmnicty Cancer Subjects Control Subjeets Tote) Enroiled

----------------

A 4 2 Causacea 57 i1 Hispanh2 23 Afftf nicanc4 5 5 Popu d eIoA e jancte ~rjet a Cnratt Subjects TetadmEnolld Age Range 2~______2 Wlanlte 3 Md an Age u a6 [035{] Figure I shows an exemplary sampe processing flowchaar tfr the isoltion of" CT[C firm collected whole blood \amnples. Fgre 2 shows the Vedrde CTC enmer~ation results Sfor alt cner samples In partcuar, thp nmber aof patients who were positive for CT~s inrasdi the later stage of cancer (Stage 4 versus Stage 3 cancers), Figure 3 provides a sutnery of HER and I R2 activation observed in CT.-os.itie samp l frmast cancrw patients acd other cancer t ypes usinsg 0e pr;oimW assay descnred herein, Example 2. Navel Method to Detect Activation ofErht am. Receptor Tyrosttne 0 Kinases> Abstrac '0351 A novel technnlogy capabe of specifically detecting phosphorylation events in Erb$ familyy receptor tyrosine kinarses (R.TKs) at a. single cell leve sensitivity has been developed. This imitxlexed pirotein microarray plattorm utilizes the foation of a imtque t. rple-anrtbod 'eaczyme- channeling" imimon~cmplex Tis principle was apied to two breast cancer model systems with limited number of rsaet cel (I) Q(Cance cells foud in a parent s whole blood: (cacuating tunmot celle, CTl~st and (2~) ancer cells found in patients f ine needle aspirate (FNA) sampl1e, This examp l ilustasthe successes. detection of activation of H{ERZ1 and RER2 (pIH ERA. and pHEFR2) in the CTC0 model system, rat a 20 sensitivity level of a single celi, and in the met astatic F NA (mFNA ) moded system. using various xeniorai um~ ocs as well as frozen breast cancer tissues with varying degree of ErbB~ RTK expres sitn, tatroduction [K521 The roei'ohip of HEWRr2 'gene status between the primary brast cancer and 25s distat nietastasis based on ona sis aftumnar tissue has been analyaed by several ~g5p(1 O5 6. Expression of a therapy tart in primary tumoer tissue may be different itom expression at distant tumor sites, and. that difference could develop over time. Loss of target expressioYn over time, wether seatmnrsrxrelated r by mtue of the natural history of disease in some patients, could Afet the efficacy of drugs directed at the target, and, if reliably known in a tiely fEashion, could be useftd in the management of patients' treatment. The dynamic namr afi cancer 'evoutior' was further demonstrated in recent wok in which good concordance between HER-2 gene status in the primary tmnor and in corresponding CTCs was shown. only' when samplIes were obtained syncthronously; CT~s fr'om 24 relapsed patients with initial HiRe2 negative primary tumor samwed thaet 9 (37%) o~f 24 patients acquired I 1HR-2 ampbtfcation in Mh CTCs (7), [0)353} nYFNA samples may be utilized to provide an organotypic and site-specific inetastatie tumor profie while CTCs could be used to detect tumor changes as the cancer progresses and therapy is coninued or modified, ThrW, re, serial sampling of tumor tissue by FNlA may be important to nimtor tumor changes as a funtion ofttime arnd therapy. 15 Obtaining reliable functional status of RTKs from singepassage NA of human turmrs would he an imporant technological advance that provides critical lufonnation to guide effee tive theraeutic decision F'N.As are .minimnaity invasive and. there fore, mnure acceptable for serial tumor sampling. Frtherm ore, the cells removed with this method can he processed within utes afte rteomovaI from the tuor; therefore, proteemic profies of FNA 20 spnecinmens likely resemble the ?n vvo prmOs very I345 The assays of the invention wideht comprisa.a uliplexed pmuxiritymediated eolabwrativemnmnoassay format aer extremely usdfA in deslig vt a innted stnmm of sample and advantageomist provide express io:Waetivation profiin of hinases and other signaltranduction pathway molecules on semialy collected CTCs and fNA tumor sarple 25 lMethods (0351 Mltilexd PoxiityAssav: ha certain emubodiments the assays of the invention are based on () a multiplexed protein microarray platform combined with (2)i antibody-enzymne clantelg signal ampliication process. The microarray platforn ofers the expandabiliwy needed to accommodate multiple markers as wed as the scalabiliy requimd 30 to deploy corcially. The unique and novel design is provided by the triple-antibody eXynye approach that offers ukra-high sensitivity while preserving specificity: (I) The selected target is captured by targetspeeific antibodies printed in serial dilutions ona 106 nicroarray surface, Then, this Rnat requires a co-ocaliation of two adtionai detor antibdi lin bked with enzyraes for subsequent channeling events per each target protein bound' (se, eg, Figure 241. (2) The wimuno-compex foned by the i aitia tgea ing by catul antoodies and the secondary binding of Glucose Oxidase ( T of congated antibodies that recognize altemate epitope on the captured target molecules can produce M 2 sz in the presence of> G ubttglucose. (31 'The target-specific local influx ot %22 is then utiized by phospho-pepde-specific antibodies coriugated with horseradish pecroxidase (HRP, TON of .10 4 !n} that bind to the phoesphoryiated peptide on the captured targets, hence amnphliong target specific sigral. Specificity for the dt~etct of I] phosphorvlated targets is greatly increased through the collaborative :immunrodetection and amplification process given the requniremnent for simruhanteous binding of three different types of anihodies. Thce detecidon and quantification of as feiw as 2- x 104 phosphorylation events is routinely achieved by this method, bringing its detection to a "'sirngle" cell level This collaborative imnmnassay configuration can be iurther applied to invetitte protein 1 intrtibons and activations. {O356@ Sjidtihitkg apture anrtibodcs were dihned in n1x PMS with detergent. A contact microatray printer (anetix) ws utilized to peti on 1 pad nroce)hilose FAST 4dee (Whatman).The spot diameter was approninaitye 7$5gn and printed slides were k ept in a desiccated chanmer at 4*C 20 (057 iEAs: Froze brs cancer tissues were from Proteo~ienex. All patients were Caucasian with diuctal carcincema at stage U or 1Li FNA samples were collected by passinga frozen tumor tissue using G.23 needle 5-1 times. Collected FINA was lysed in 100 l p lysis buftfet and resulting samples were stored at -80*C until the performance of proxumity assay. f(*3583 K1gg tunior THuman breast cancer cell lines of MD)A~MB4S. MDA-MB)23!., 2$ and BiT4 74 were subcutaneously injected into nude mice, When the tumor volume reached 400 rnuW in size, F'NA samples were collected using G23 needles, Collected FNA samples were processedi as described above. jt)3$9] Se~itii We detected the activation and expression of HERI and HER552 at a 30 sensitivity level of a single cellin mulitiple ell lnes $4MPMB468. A 3lI 3424, and SKar4 cell ines). These cell lines express I 0eIO total tKs on teir ell :nembnratper cell. although only subsets of the total RTKs get pbosplhorylasted and such phosphoryitamo is 1.07 requrd to pathway activation. The SKBR -3 cels hae spontneous 'HR2 sctlysfoa Oue to ihs amplifiction. and therefore they provided a positive control reference, MA MB-46g cells need to be stimulated with EGF (TGtF) to induce llER1 phosp orylaioe and their srature before and aller simuladon caSn be used as negative and positive comrol MDA 5 MB-468 has marginal VR1. activation before stiititon, while th cel hie peak at approximately 10% of their .RTKs activated l" 1 x I0 hospMhryltion events per cell), Our assay tomtat. enabled us to detect iess than It? ttctivatton evens with 'gle en siiiy a snown in Figure 4, 0360) Xenokuraft-FNA: To demonstrate the potential applicaon of our assay in milNA 10 samples, we first developed xenogratt models for difehreat types of breast cancer ushtg cel lines with varying degrees of ErbB-RTK expression (VIDA-M-23,1fDA~MB43$, and BT7 as shown 1 Figure 5. We detected l w levels of pHF R2 and pHE1 in MD-M1 -23 1 xenogral> As, signticant levels of pHER2 in FNAs obtained fom BT474 xenog =as, and very low HR , or HfERS activation in FNAs obtained from M)AM45 xenografls. Our 1$ findings front the xenogafFNA model system are ow ondant with the driver ceidiae F1ER2 proille demonstating that this method can be osed to detect activation of ErbB receptors in samples obtained from minimally invasive procedures such as CT1C and FNA of beat. cancer and other types of etastatic cancer [03611 FzeninevE\: To further demonstrate the utility of our assay ormat we 20 collected FNA samples from 2 a to Ufozen breast ducta carcim (14.wih kn HER2S lEC status) using 023 needles. The activation of' HER2 receptor detected by or assay is concordam with the tumor UHC score (Figure 6), We have 4 patients with high 1HC score (3) in primary turs. All of them have high activation of tHER2. interestingly, one of 4 patients has high activation of hot EGFR and HER2 receptor Tis indicates that a 2$ therapy of T1(1 inhibitor msay be more etbective than Hercrreptin alone, 1W362] Figure 7 (left) provides a sum of the levels of activated HER I ndE i FNA samples from breast cancer tissue with know c or unknown HER2 1HC status and normal tissue. Figure 7 (right) provides agraphical illustration of pEGFR snd pHEiRZ levels in FN A samples with unkinowni HER 2 11HC status, Figure 8~ shows a titration analysis of 30 pHIER2 levels in ENA satmples with high 111C sores (3±) Sample iD Nos.tl$$12 5 ad 0 1 1097 12). Fgre9 Xshows, th detecto n of pOlt an 1 lER "R vNA saples at w dierent time points using a seral dilutior of four ditrrent capture antibody concentration I 8I 1%331 A novel technology capable of specifically detectig phosphoryiation in ErbB fatrily receptors with sensitivity enabling use with myte C'TCs was developed, The exprsskma ctivatm profinng af kinases and other sinal transduction paha mnolecu les on S a serial sampling of CTCs provides valuable tntrxnaton on ranges occurring in turner cell as a function, aof time and therapies. This therapy geligdiag anstic can be used at various stages of the disease management, as sown in the Figure 1,t Because ofts unparalleled sensitivity and specificity, our approach can be applied to detet phosphrylation events i Irb3 family receptors in. are circulating tumor cells (CTCs, By identifyig HERi and 10 HER2 activwiion in CTCs and FNA samples, this method can provide guidance, niot only fort initial selection of targeted therapeutics, but also in. subsequent rnuonirng for rapidly evolving' cancer signatures in each patient, t4364] As such~ the rnultipl exed proximity based clabaratilveinnoassay platform 0f the preseniedro pmoid alable cainical infomation on linied aplers ac astICs 1$ ad mfPN~s wthultra ensitivit and specifieity to aid or assist oncologists in adlusthng their dsease teatment oons beah patient accordng a &persona< cancer prfieshf lopisoieasefl inPrmar ad Metastatic east Cancer. Cancer Resarch G 134 20 3411 2.0C $36pln vi Wgnr Rend Schorn gg Rt Bevda Ndoony H. inr P, 1-erzog BI ascud A.Zlobec I Q Cahomas G1 Terracciano I> Pchier C Bubnder2 11ER12 gene status in prinary breast cancers and matched distant mtetastases eaTh'" ancer Research 9.R 1.2007 25 367] 3 Lear Kaal Kt Yon i Kleinsebnritiehiasters B3K Mciavuan ngh Mio li H 7 +/t n atu a 0 reastCancer ietastasestthme (centrNervos Sy nstem.AchPi ab Med 127. 14 14=5 200 0*368] 4. Sinmmons C Midhr .N, Cuddae Qanfe(i D Oif xCdield Mi Draitsadies CG dimnsea Does contimatorn nor biopsy alter the management of breast ergatients 30 with distant metastascs? Atn ofOnology, doi0 i / anno .adpO2, 200 20 5348. M0 [0369 5 FfaHiA lnedeto A Metm Q Mehlced IV, WI P stico ussil- Mf, PaatAgottoCee Changesin HER over passimbetweenpaatrynmor and atologo usnntites Caurxeatienswith cihicd andt biological tfeitttues b0370 6. Meng S Tripathy D, Shete S, Ashfaw R, Sabuoran H, Haley Brekel E, $5 Eus f, Leith 4, Osboran C:, Clifford E, Perkens S B.tsch P, Khan A, Moron L Herlyn Dt Terstappen LWY Lanc N, WVatn S, Urbh uPAR and HER- gene stats in individual boast cancer calls fmm blood and tissues. PNAS 103:1 7361-4, 2.006, {03711 7. Meng S Tripathy D, Shete S, Ash fw R, Haley ID, Perkins 5, Beitsch P, Khtn A, Buhus D, Csbome C, Frene E, Hoover S, L-eith . CIIfford E, Vitetta l7 Morrison I., 10 RerynDTerstappen L Fiemming T, Fehm TF Tuke R, Lane N, Wang J, Uhr . HER-2 gene amplification can he acquriredt as breast cancer progresses. PNI AS 101:9)393-ft 2004. Exmnple S Profiling of Receptor Tyrosine Kfrase (RTK) Activation ini Credating Tumnor Cells (CT~s) in Metaftatic Tnrs Using PruimirityMedhated Mirrarray iwutoasav 15 Abstract [372g The abnormal activation oI EGER and HER2 has been liked to various types or cancer progressing, and the changes in. expresson status between primary tmor and CiTxs have been reported to oczur at a signin ant f&crequene Methods for' detecting E0FR/HE11R2 phosphorylation n serially collected CTCs may povid' vai able iN Sight into the overal 20 disease protfile shift, and ilverefore lead ti better weleti on of therapy conmbiaion lox individual patiems. A riple-nibody-ezyme-channin ltipdexed protein microaray platttonm wats developed to detect the phosphorylationa n target nmolecules, .! utilizes a uique immuOomlex trmtation 'via co-ocalizaion of two detector rn enjgated antibodies once target proteis are captred on the mirarray'surface. The channeling events between two detector enzymes in proximity enabled profiling of the RTs with a single-fel level sensitivity, In order to validate the method on clinical sample, CTCs r 27 breast camera patients wit nmetastatic disease on aious dherapy regimes were analyzed The nulIplexed-proxnity mediated immnoassay successful detected the amplification and ctivation of RTK in CTCs isolated fro various cancer patients, CTCs with amplified 30 and activated HER2 were found in 5 ot of 17 (29%) breast cancer patients with HER negaie primary tumors. As CTCa found in the metastatic stage rep rnt the most 110 aggressive inj ding aCellpopCLatiot serial iCpriiding ntend to better thepy seet n/ad usimentand disease monitoring 11ewt the V0 1 nlt [03731 There have been numerous studies detection of carcinom celts in the bhod of patient with solid rmnrs. Detecton of creulating tumor cells (CTCs) before initiation of irst-ine therapy in patients with metastatic breast cancer is highly p active of progression tree survival and overall survival (~3). In b onship of HER-2 gene status between the primary breast cancer and distant metastasis based on analysis of tumor tissue, has been described i a number ofstutdies (42 0), Expression ra. 0 therapy target in primary tumor tissue may be different from expression a" distant uorie sites and that difference can develop over time. Changes in target expression could aiTet the efficacy of treatment, adifrlbly known in a timely fahion, could be used to guidle therapy, As they are more readily obtained from patients than scrial tumor biopsies, CTCs (as well as FNA samples collected from metasttie sites) may be used to monitor these 15 changes, The multiplexed proximity based immuno assay described herein is used to the expression and activation levels of the ErhB1 family receptor tyrosine kinases (RkTKs) EG R and HER2 in C:TCs. Method [0374j iviihtexsirringit Arsas: In certain embodiments, the assays of the invention are based on (I) a multipiexed protein microarray plaOrmI combined with (2) tripe untib'odvrcuryme chasmeling signal amoplifi cation process, The unique andt novei design is providd by the triple-anrtibody enzyme approach that confers l tra-igh sensitivity while preserving specificity (1) The selected target is captured by tinget-speetic antibodies printed in serial dilutdons on a microarray surface, Tis format requires a co-ocalizaion of 25 two additonal detector-antibodies linked with enzymtes (see, eg, Figure 24), (12) The inmmuno-cmpiex fomed by the initial target binding by capture antibodies and the secondary binding of Glicose Oxidase (GO, TON of I U'7mia) cornugated antibodies that recognize alternate epitope on the captured target molecules can produce Hcl:O2 in the presence o O substrate, glucose ) The targe- e ocal influ f 2 30 utilized by phosphopepide-speciic antibodies conjugated with horseradish peroxidase that bind to the captured larger, Specificity for the detection of phosphoryated targets is greatly increased through the' requirement for' simulitaneous binding of three dilTent antiboxiies, Ihe dtoecton andquantifcan of astw as x 0& phophcrylan evn s nrUiUnid achieved by this mtethos d ing it detecton to asing cecl level, O375 Slde tig A contact croanmy pointer (Genetix) wa used to pit onil6pad ito (:ose 5A;sdes (Whann-n) [0ff376) FNAsatoenbreast cancer tissues were tfo P.roteoGenex All patients were Caucadian wth ducta hr 'rst ca rcinoa at staged Uor Rl FN4A saupl es were colleted by passing fmozen tumnor tissue using 023needle 1~i0 timier. Cdiected H w rs lysed ad stored 08 at -SWati h pedinnce of proxinmy assay 19371 Si fCs were isolated frontwhole blood of cancer patients bytnag-netic 0 prutiels coated h ati CAMatibodes usg e C o de. nid CTs wereav atedgsed mdstored at 40 C unti peroace of' the assay Results PU378 eQsitVy: We detected the activation and expression of iGFR and KER2 eta senitivity level of a single cell in cell lines A43I and SR13, These cell lines express ~ . 1$ a 108 total RTKs on their cell membrane per cell, although only subsets of the total RTKs get phosphorviuted and such phosphoryltion is msuired for pathway activation. Both el lines peak approximately 1 Os of their RTKs activated (~ x I03 phosphoryhation events per cel), ur assay format enabled s to detect less than 1W acaiation cyxnts with single cell sensdtiv as shown in Figure I1. 20 (03791 enTisueFNA: To Iurdher demonstrate the ttity of our asay fonat we colectd snples $tn 29 stage i to II fi'rzen breast ducial carcinoma (14 with known 4HER2-IHC status) using (23 neede. The expression and activaton of H ER2 receptor dected by ou rs i conordant with the tuor 114C score (Figure 12) We have 4 p n ho a hgh 1 H C soxreni (3+-i) is im tonrs, an 1o! 4 have high expression and 25 high activation F E. iterestingly, 20% of R positive patients with H ER 2 expressing (+ 1TC) have a fair amount of activated HERP2, 'hus could have implications in parties resistant to horttxnl themapy, {M380 F re 3(tap) shows a "hear ma' iliusmaing jh levels of -hobphorda bih rceptors 1 R(2 )IER2, E ad p95 5 3K, and SEC foni 12 Wthe N a o0 now HRz T-I sai. fhc daahoninthi fiue ntnathattheactivatio ofHEIR receptor detected by priity assay format i concordant with the pttaty teer 1O 112 cnxe. flgue 1hottan) shows a Wes tam hot analysis o te{otAWER2 and p95 levea ubset of the FNA amls' wih known HER2H aGtatus. j9381j EfS: C1 Vuwe ilated fromT the blood of patients with metastatic cancer, In particular, ANs the whole blood of 27 metastaic breast ennee: patients and 60 healthy 5 volunteers were analyzed for EGF R (IRI) and lER2 expression and activation. The niunber of breast cancer patients with negative H ER2 expression in the primary tumor and conversion to HER2 positive in CTCs is shown in Figure 14, huportantly 2% of patients with 1HER2 negative primary tumors hadi CTCs with amplifled and activated HER2. Fgnre 15 shows confirmation ofUHER2 expression inC CCs by lHC itnaging' (Veridex), .0ff382J Lire i Rel"ctln: Based on itsing of 60i healthy controls, lower limits of detection (LOD) and Inower linbs of quantitation (L LOQ) were determined for total and phosphorylated IhRI and RAER2. Data are shown La Table S in cmputed units (CU) based ona calculations fhrm standard curves generated with cell lines expressing known levels of HfERI or HER2 and the degree of phosphorylation upon stinutation. LOD LLGQ4 EGERP 0,693 3,20 HER&P 9,25 0,51 EGERT 53.06 80,0~ HER2T L.75 3 20 20 Couchwio [0)3M3 A novel technology with unparalleled seni tivity and specit ci ty successfully detected the activation of ErbB RTIKs in CT~s isolated fro c nce patients. Activated hEFR and HER2 were detected with singl cell sensitivity, Testings of FNA samplies from frozen breast cancer tissues showed concordance. between reported UHER2 status (1MC) and 75 results with our proximity assay. The expression/activation profiling of kinases and other signal urnsduct pathway mecunles on a serial sampling~ ofCa provides valuable information on changes occurring in tumor cells as a function of te and terapies. This method can provide guidance, not only for: initial selection of targeted therapeutics, but also in subsequent mronitoring for rapidly 'evolving' cancer signatures in each patient by 30 nalyzing rel evant but limited amount of samples such as CT~s, metastatc .F.NAa, bronchial lavage tlid, and the like. 151 [03841 Importantl, this method enables the idemidcion of patients in which chages between primary tumors and CT~s with regard to the activvatiotn and/or expression status of certain EbB RThS such as RER2 have accurre Fr example. CTCs with activated RR2 were found in 29% of metastatic breast cancer pate'ts withER2-negative primary tumors 3 demnonsating the utility of the method described herein~ fbr detecting the conversion of iER2-negative primary tumors to H1 ER2-positive CTCs and for guiding treatment decisions (eg, Herceptin therapy based. on detectn 'of HER2-positive CTCsY As CTCs found in the mastti stage represent the most aggressive invading cel populationx serial CTC~pro filing can lead to better therapy 'eecin/djsmetand disease monitoring. R0feferenees [038f5 L Cristofaniui M, Budd (T, Ellis MJi Stopeck A. Marera 3, Miller MC, Reuben JMH Doyle GV, Allard WJ, Thrstoppen L, Hayes DF, Cireuhiting tumor cells, disease progresson, and survive in meastatic breast chance N Eng- J Med 351:781-91, 2004, 103S61 iethdorf S. ric ,M l V, Rau T. Schindiheck CR Rack B, Jianni W 15 oth C Beek K, Jaiekie V, Jackson S, Cornet T,' Cristf l M Pantel K. i of circulatng tumor cells in peripheral blood, of patients with metastatic breast cancer: a vawdtion study of the CeilSearch system. CGin Cancer Res 13: 920-8, 2007 [0387s 3, Cristofanilli i Broglio KR, Guamer V JaksonS, Frtsche HA, Islam R, Dawood S, ReubeniM, Kaum SW, Lara J Ki nmhy S Uae NT Hobagyi 0N 2:0 Valero V. Circulating tumor cells ienimetaslatic breast cancer: biologic staging beyond tmor burden, Cin Breast Cancer 7:471-9.2007 0(3884 4 Tarned I, r'wninen P ando ain Aipuication of l'hE 7 /net snd Topoiscnersee a n ThnarK and Metnarc. Brea Cancer Cancer Re5earch1345 z348,1100I, 25 P3891 p Uai Sato S. Wagner U Ren Schaegg R Hed Novoty Grili Hierzog M4 ilaasud A. Zlobeed1 Cathomnas Li ehrraccianoPeiTechter C, iubendorftL. HER2 gene status un primary breast cancersanmed e distant metastasea. Bretst Cancer Research R31. 2007' 96 4 tear-Kau KB oi Kinschmidt-DeMaster BK AcGavran , Sinih M! 30 HER-2/ueu Stuns in re a ncr Metastases tohe Cental Neos System Arhatho Lab Mcd2 45145 2003 0391 7., Simmns C, MiEWr N, Geddie W, Gianfelice D, fel M4, DraTaries 0, C&enmons M, c Ds cormatoay tuwo bipsy ate the man aemnt of beast cancti with. distant metastases? Ann of Oncology, doi: 10.1093/ anno no/ mdp028, 2009. (0392! & Fabi A, Benedeto A, Meir G, Melucci Ei, Vic P. Nistico C, Fusillo M, C (ogetti F, Mo IleeM Changes in HER12 overexpression between primary tumfor and autdogons nmtasases: Coreatios with clinical and biologi&a theatre 10393I 9,. Menog 5, Tripathy D, Shc: S, Ashfaw R, Saborian5 M aley B, Frankel E. Enhus D, Leitch M, Osborne C, Clifford E, Perkens 5P Bitsch P. Khan A, Morrison L, Herlyn D, Terstappen LW, Lane N Wang J, Uri uPAR and HER-2 gene status in 10 individual breast caacer cWlls from blxoi and tissues. PNAS .103:1736 -4 2006. 039 0.4 Mong: S~ Yapahy V *Shote S Ashfaw P. Haley 1 Pedains S aisalh PKi/han Rx uhus H, Osbomne C. Frenkel E3 Hooter 5, Laitha M4 litiord FVitetta E, forrisonL heriyuD TerstappeL imning Fhm Tucker Rl eameNangL : HER~2 sene .amnpiicatiorn can be acquixedi as ireast cancer progresse. .PNAS 101:3934- 204> 1$ P395] L Puszta L, Ayers M, Ste , Catk E Hess KQ Sivers D, Damokosh A, Srige N icbhhbA TA, steva Fl Arun B, Cristoanilli M Boo sei D, Rosales M., Valero V Adams C, Hosobagyi N, and Symmans FW, Gene Expression Profles Obtained from FinNeedle .Aspirations of Breamt Cancer Reliably identify Ro utmne Pmognostic Markers and Reveal Large-Kcale .Mclecular Differences between Es'roge-ngative and Estogeno-positive Tumors, Clinical Cancer Research, Vol 9, 2 406-2415, Jy 2003, -Example 4 A4n, ys of irb Famly Receptor Tvrosine Khase (RT7) Acivation in Herrceptia Sensitive andi Resistant Cells, 10396] This examples ilustraes the analysis of ErbB activattn and dimer foation in herceptir seusntive arid resistant cells, lit partid ar, this example shows that an increased 2$ ieve; of actdvated p95/ER3 heterodimer is associated with resistance to Herceptin. This example also shows that highar levels of activated p95 arnd 111RR12 are itse il erceptin resistant cells. Finally, this examples shows that Heorceptin sensitive celfls have lower levels of activated p95, THR2, HERS and P13K, As such, the effacy of Hrceptin treatment can be evaluated and optimozed by detecting the presence or absence of any changes in activated 30 p95/HR3 heteroditner levels and/or activated p95, HER2, HER, and/or P3K levels, This 115 method advantageoslg leads to a more $atwned therpyseletiondi stent and disease monatorig; [0397] ilcreeptin resistant ceOW lin' (BT/RI: The cells were cloned from 'T-474 cells. erceptin does not inhib UER2 phosphorylation and c m growth in BT/R cell BT/R and 5 BT-474 cell samples were treated with 100 giii of Herceptin f2 2 8, and 24 hour. The cels were iysed and the protein concentration determined by BCA protein assay. j0398J Figure 16 shows hat there was a signing inhibition of phosphorylation of i11ER2 in Hlerceptin-sensitive BT5424 cells upon Hderceptin treatment compared to $1474 cells without Hterceptin tretment. Figure 16 also shows that Herceptin-resistant BT>1 cells 10 displayed signi ficantly~ higher activation of RER2 compared to Hercepmtin-sensit'ive BT5474 cells upon Herceptin treatment, Figure 16 fuher shows that there was a moderate decrease in total HJER2 levels for both Bl474 and BT/R cells compared to the same cells without H er1eptin treatment (0399s Figure 17 shows that Herceptinresistant B/R Cells showed signinmicatly higher 15 activation of p95HER2 compared to Herceptin-semitive 174 cells upon Herceptin treatment, Figure 17 also shows that Hi eeptm-resistant BR cellI displayed increased activation of HR2, HER3, and P13K compared to Herceptin\sevstive BT474 cells upon Hercaptin reatment, Figure 18 shows the expression of HIER1 p hlR2, M4BR2, HER, P13K, and SHC in both BT1474i and BT/R cells treated whh Hcecptin, 20 W0400N Figure 19 shows a schematic of the ErbP pathwvay in BT1474 cells in the absence' ot Hekrccptim UER2f and HER2/3 dimers were detected. H-ER1/2 duimer was very west. p95/HER3 dimr was 3-4 1o d weaker than in tR17 cells [0402 Figr 20 shows a schemati ofEA pbwa' moduladioni iT7474 cels with Noeepti teatment HER22, FER i 1and H-ER2/3 diners were down regulated. There was 25 no change in p9$/lER3 levels [t41324 Figure 21 shows a schematic of thc PrbBP pathway in DT7.R calls im the absence of> 1lereptin. H ER 2/2 and 2/ dimerm wre 2~3 dimes stonger an in BT3474 cells. IRR L/2a dirmer gae a weak signal pllRER13 was '- i'oid stroger than in BT1474 cells. 0403 A gure 22 shows a schematic of Erba pthway modatin TR cells with 30 1-erceptin tram t, HR2 dimer increased at the 2 hour poil and thn wem down. 110; H1ER/3 dier was dovsnnguatedHI A dimer was downr elated. u $/1ThI3 increased and then stabilied. Emple 5. Aae~ ry Pro xinay Aasay Sde Fortnat. (414 This example illustrates one preferred embhodiment of the prmiyassays of the present invention, The prQxmity assays of this embodimnn use an atibodymicroatrray based piatform that measures the expression and ativation of taret proteins in circulating tumr edIs (CTaS) and/or tissue samples (ag, FNAs). The proximity assays analyze the level of tein expression and the status of salvation by analyzing the degree of HE1 and UER2 phosphoryiation, In some instances, the proximity ssays of this embodiment utilize 1 CTCs isolated from about 5 nil of whole blood by magnetic paricles coted with ant CAM antibodies using the CTC~Profiler (Veridex), Iolated CTCs toay then he stimulated with growth factors (~gg EGF ± eregulin) prior to immuno-ainalys for the subsequem lErhE pathway expresin/activation, {04=1 In certain instances. the proximity assays of this embodiment use a slide Iormat and is include dtiple calibrators and control Figure 23 shows the array designs of exemplary s~le ormts fr analang total and phosphorylated HERt and HER2 levels, There are 16 piad on each slie with room 2 fo 300 spts on each pad, A contact microarray printer was used to print on the 16 pa a illulose slides. Each spot includes a raking dye and either specific capture antibody (Ab) or control pri ed in tpcat serial dilutons. The 20 capture Abs ee printed at i mgm, 0,5 mg/mi, and 02 mg/ndL Purfed IgO was printed as an oientation reference in both the Total and Phospho aasays. BSAphospho was printed as a reagent control, Analytical caibration reactions are performed on pads and itemal quality control reactions on 2 pads, Eeh slide allows processing atfup to 4 unknown roatient sasnpfes. Expression of total target proteins or phosphoryhtedx activated proteins can be 5 reported in Computed Unit (CU) a unit based on calculaon n fiomstandard curves of diluted lysate from positive cell lines which express the protein o, Two s ate sales are used lor each sample; one slide to detect the explosion of the target proteins in cells isolated from whole blood ("Tota. Assay Slide") and the other for the detection of phosphoryation to detect the degree of target protein, activation ("Phospho Assay Slide"), .0 4I06 in this embodinuntn whole blood from patients an4 normal control ndividuals are cooited inITA tubes. nn rde to prevent any skin eti contamination during blood draw, our procedures stipulate that thefst 3 mL of hiood collected isdiscarded (or ollected in 11; Cell Save tube for CTC counts arnd visual immnuno-staining using CtelSearch kit). Two aditionl EDTA. tubes are ten used to collect 7.5 L of' whole blood in each tube, CTiCs are then isoated from each Mhe using an automated i metic cdl e t vice ( dd AutoPrep), Enriched. samples are combined and then stimiulatezd with gowth atrs S Activated cells are then lysed anid either immediately processed or stored at ~804C for subsequent immuno-aialysis. W0407] The proximidty assays of this emsbecdiment are initiated by incubating protein talrgetts in stimulated Mtl Wystes wih captre antibodies on an mntrro-microarray surfae, Any IERI or HER2 RuTKsi cell lysates are bound to their corresponding capture antibodies and 10 subsequently unique mxnmno-complexes are formed by two additional detector antibodies, One of' te detector antibodies is conjugated so glucose oxidase (GO) and generates it in. the presence of' glucoe;, When thre second H-RP-contjugsited detector antibody is bound ini proxzimilty within the immnroo-eomplex. a positive signal is generated, The subsequent tyramidemediated signal amnplificaicn process enhances the sensitvity of toe assay. The 13 specificity of protein detecmion is enhanced b) the concurrent bidding of three specific Abs to differt eopes, ad senstivity ca be as hoo a single cell due to the ampyiication, Figure 24 shows a schematic of ian exe mplary proximity assay for detectig phusphoxylated IlER 1. A48 'The mnicroanray platiforn described hereini offets th enefi o nutipieing Thea 2t abi to expand he asay snbies hgh contt andysis Wh the msureinute o nie receptorn and signsin a moecuiles fom limited available sample. The rncoanav is scalabde and has the potential fat achieving the thr ghputneeded for a cialldy rscfdiagnostic assay. Example 6. DetectiCa oftrumetd form of HER2 receptors and other receptor tyrosinc 25 Mases using mierarray inum anoassay, PD4091 iER2-overexpressing breast cancer has pooter prognOsis and is often resistant to HER2 targeted monoclonal antibody therapy, One of the mecanisms of d novo or acquired resistance is expression ofp95HER2, truncated HIER2 receptors 'wih missing amino-termna extva ceullbr domains. Methods for protilng various forms of HER2 receptors and other aua receptor tyrosine oases (R T) with potential to fbnn hetero-driers for their level of expsression and degree of activation on primary and metastatic mors provides valuable insighlt into the shifting disease pathogenesis, i i (04 4 Methods: The tinhnoiogy described herein can specificay detect phosphorylation events in Erb family RTKs The Omltiplexed protein merCrray phitform utilizes the fomain a unkque immusno-compieu equirig the c localization of two detector enzymetconjugated-anatibodies once target proteins are captured on the midcroarray-swifce. S 'Thet chaneling events between two detector enzymes (glucose ox idase and horseradIah peroxidase) in proximity enabled. the profiling of the RTK with extreme sensitivity. The analytical spcificity is greatly enhanced given the aquiremient for sinmtianeous binding, of three ditferent antibodies, Diffeent configuration of detector antibodies allowed differential detection of vtacated targets (exg., p9$ER2) from their normal center parts (g. IER2) I) Sucessful profiling of signal transducton pathway molecules tot thir expression and activation using 240 FNA sap le collected foi breast cancer patients (stage Ii to IV) with various ER/PR/RER2 status is described herein. [041 1f Rtsut The FNA samples collected using G23 gauge teedles were analyzettor expression and activation stats fior various RTKs including p95IE2, HER2, HEI1ER3 15 P1K, She and TGF-IR. The results indicate; >- 100% concordance between pdrmary H{ER24H C status and HE~iR2 expression > Presence of varying degree of p95HER2 in over 40% of RR2-positive (HER2: 3+ andI 2+ h FISH+) patients > ~50% of p95HER2lt expressors had activated p951i ER2 20 > 25% of HER2positie samples also had some levels of ER 1 activation > HER2negative (E2: 2+4- E4 FiS, i+ and 0) samples also had 100% concordant ETE:R2 expression, but a number of them showed low but ignificant levels ofl'HER2 and HER! activation. 'These find ings can he used ar section ot approirdate tatieted therapeuti 25 (0412] Disension: The uliplexed-nnmuno microarray was utilized to detect the expression artd phosphorylation of p95ER2 HER2 other RTK (including HER3, HERfL IkR, S$H4C, and P13K) in 240 FNA samples collected from unique breast cancer patets with various ER'PRtH}ER2 stares. Having te ability to profile tumors at different metastatic sites with an expanded pathway panel cud provide inIermation on their difTreta Al 3 mettatic potentials; hence minmAll tnvsive single-passage-mFNA sampesa be utilized to tailor therapy options as diseaseprofile changes, Examiphle7 Profiing ofteenc~rrent breast cancer patient% for their Erb$ pathways using circulating tamior edis for therapeutic implicatin. P1413] HRR2 is on e o u tranembrane receptor tyrosine kinasez (RTK) in epidermna. swnsanCrni n growthLIT fact por f ya ER2-positve phenotype has been associate with > 5aggresive Subtype of breast cance with HER2 gene amnplificatiorm Approxitmately I 5 to 20% of breast cancers are considered HEfR2-positive by IHC or PISIL analysLs Changes in H1ER12 expsression status between primary tumor and circulating trumor cellis (TCs) found in recurrent metastatic disease have been reported to occur at a significat frequency, Methods for detecting HER12. exprerssion arnd pihosphorytion in serially collected C<Ts may provide 0 valuable insight itao the overall disease pnfAe shift, and therefore lead to better selecion of therapy corobinatlOn for individuals patient, W04141 Methods: A trie-antibody-enye-channelinug multiplexed protein miceroarray plattenn has been developed to detect te phosphorylation on target molecules. T Cremely high assay specificity was achieved by imnrmo-complex formation via co-localition of two bg awa -0"5'' 5 detector enzym~e-conugated-antioies once target proteins are captured on the mcroanary, surface. The channel ig evet s'etzen two detector ernvymes in proxinity eabled proflin of the RTKs with a single4-ell level sensitive ity. 1t order to validate the method on clinical samopler CTCs fronm 77 breast cancer patents on diTerem therapy regimens were analyzedat varions time points along their course of terapy. 70 [Q4151 Reults CTCs from whole blood of 77 metastatic cancer patients and 6 healthy vouteer wr'malyed for HiR2 expression and activatior. Sionificat HER2 status conversion with recurrent disease was observed, 29% ofpatients with. negatve H ER2 exrssion in the primary tumor showed HIER2 ivmpliincation in isolated CTCsm. Phosphoryhed HER2 receptors were found in 52% of patients with primay HER2 negative 25 disease The enhancement of assay sensitivty and specificity using proximity mediated nimnunosassay made the detection of HER? activation (even without anmplifcationl pcssie whon isolated CTCs were stimulated with ligands. P0416] isc ussian: 'The multiplexed-proximity mediated irmuo assayv successful daeected the expression of HER20 RTI.s and their degree of activation mn CTiCs isolated from recurrent breast cancer patients, CTC~s found ins metastatic stage represent thre most agressive and invasive cell population. erial CTC-pro fitlng loads to better therapy solection/adjustment and diseas/tmatmrent monitoring as there are available options to C".121) chose appropriate tinase inhibitora fo RTK.targeted therapies. While a significant number opa s amired HER2 ampliication in ther C' Cs, a s ntialiy higher rate of CTC HER2 activation was found in relapsed metastatic disease. The unique tripleatibod y mediated immusno-microarray anralysis allowed a single cell level spoiling of the CTC HE1VR2, This principle is further applied to profile expression/activation ot'othet signal transduction pathway molecules, The ability to profile serilly collected CT~s provides valuable itrmation on changes ocringn tumor cells as a functio n of tine and thraies. This method can provide guidance, not only fr initial selection of targeted therapeutics bu also in subasequent mnitoring tor rapidly evolvingg cancer signatures in each patient 10 Examnpk 8 Chrtratrfata at' f Hrepthr rmistanica using hmmurnrandecroarray baseri pathway analysis. 104171 Background: R x breast cancer has poor prognosis and is often resistant to HE1.R .'argeted mnonoclonal antibody therapy. One of the mechanisms to de neo or acquired resistance is due to the expression of p95HER2 and/os by formng heterodimers wth other members of the ErbB receptor tymine kinase (RTK) famiy. Cancer cells ajso tale advantage of signal tranisduction pathay redunodaney and fom heetdimners wah neon ErbB RTKs such as 'G. R, This examie describes the characterimion of a mechanism Herceptin resistance using HER2,anplifidd cl ines with different sensitivity to antih-iER2 monoclonal antibody therapy, Breast cancer with HER2 amplification should be further sub~ 20 profiled for other pathway molecules to identi yt most effective targeted drugs, j141 81 Methods; A novel technology capable of speciicaitly detecting the level of expression and the degree of phosphorylation in Erb finally R.TKs 'eg, p95HER2, HER2, HER3. and HERI).bsequen downstream pathway molecules (eg, Sb mand PI3K a non-ErbB RTKs (eg W.1AR) has been developed. 1Ts mutiplemed prtain microarray 25 platform utilizes the fortnation of a unique nmmno-ceomplex requingmO the ro localization of two detector enzyme-'conjugaited-'anti bodies once trage proteis are captured on the nictoarray-'surface. The channel ing events between two detector ernzymes (glucose oxidase ad horseradish peroxidase in primity enables the pmfibng of the Rkis with extreme sensitivity, The analyt eal speciu fiety is greatly enhanced gienth requiremtent tor 310 sim uaeous binding of three diferent andbhodres. Different configurations of detector antibodies allow differential detection of truncated targets (eog, p95HER2) fom their nomal counter parts (eg. HER This exarnple descrlbes th differently patten of p95HER2 associated (eg, p9HER(2UIER3 heterodimer) and on-associated signal transduction 121 pahway nolecules including p95HER2. HR(2. HERE H2R3 P1K1 Sh, and IGiti R upon Het tepa tfletm in - sE tRampited cels with different: -targeted * k ditmto2j ght1010.f rsxhr~ eidt to HIIQk mann anaw antibody therapy S * ~Hercepthmrteistant Ei474 cells showed an approximately $t a stronger p9$UERP * Hm"Ph unrseen mdscndUm lava! l 1 ediated pathwaiay and vo, i an e ep didsens'topuilat owhen reattei with iieoepti * In addition to 2 to 3-fold higher levels of phosphorylated HER2H and 1HR L weak I0 IR activation was also ound in the Herceptin-resistant cell population 10 * Heceti treatment :rdoced the level of pbosphorylatioo aong hER2 and e1Rm 3 R TKs itn Metrptin-.sensitiv cells, f0420] DiscussiumThe matipkexed lh immr tYmicroatra was utilized to detect the expression and phosphoryiaion of p 9 $HER2, 4 ER2, othe RTKS (including HER3 HERi, and GF,11R) with the potential to form heterodimers, and downstream pathway molecules 15 (including Shc and P13K) -laving ea remiey high specificity and sensitivity due to its intnsic triplerx- mn.assa fotmat it has unique potential to be utilired on limited amounts a sap les (etg, CTCs and FN As Serial>y collected stupis can be treated with potential targeted drugs and post-treatment pathway analysis could be performed to provld msechanistic insight into the lewel of ecffectiveness of such u attment~ This platfor pnwisdes 20 ieeant clinical information to select the tuost efetive therapy and monitor the adnunristered Example 9, Charactriza tion of Weeptin re4sitnce. length and truncated forms of HER12 (e., p951ThR2) has been dedoped. IHere we report 2~ characterzation of a mnechanismt of Herceptin resistance using LliR2-amplified cel lnes with d(hfferent sensitivity t~o ai-R EilR2 monoclonal antibody therapy> Diffenential activation HERB2ape ce\h upon. H erceptin. treatment are reported Hereptin-restanxt BT474 edla shwe sinifcntly higher phosphorylation of p95HEfR2 than H-erceptin-sensitive cells upon 3) treatmntw wit H rin while expression of total HER112 and p951HER1 2 decreased in both sensitive and resistant cells Analysis of pathway activation and potential mechanistms of 122 rsistnce such as expression of acti vted p95HER2 are used to select targeted therapies most likely to beneftt a pasticular patient ad to monitOr response othrp ve ie [tM422) HERf;2 is overexpressed in appnxinateb 25% of breast cancers, and the ovrexpression isa ated with a poor prognosis. Targeted therapy with IER2 targeted moonocional Ab (H-erceptin) provides substantial cliical benefit, H'owever, many patients with HtR2 ovaxressng breast cancer do not respOmd due to he rvo resistance or acquire reistance to Herc eptin over time. Multiple potenildmecbanismis for l-erceptikn resistance have been described, including activation of I 1, activation of o-MET and inactivation or loss of PT&N, Another potential mechanism of resistance is expression of truncated forms 1 of the HER2 rceptor which act etace uiar domain but have kinase activity Truncated ER2, known as p95111R2, is exrse naproximately 30% of breast tumors ovteexpressing RER2 (Scariti 20071) p95 can heterodimerizo HER3 and actvation with hereguli induces p951ER2 phosphoryation. Activaion of p95 can be inhibited by tyrosine kine .inhiibitors but is not inhibted by Herceptin (Xia 2004)> The presence of high levels of 15 p95 in primary breast tumor tissue has been shown to a strong prgnost tor, marking a subset of H-ER2-postive breast cancer patients fort worse outcome (Saer 2006), lHere we report a baracterization of' a meehumism< of Hterceptin resistance using HER2-k1ampified cell lines with different senssitivity to andi-HR2 maonoetonal antibody therapy. METHOD 2> {t42 Anovel technology capable as spec lica ly dettctntg ihe evel ofexpression and the degree of phosphorylition in Erb fauy R.s ( p95HER2 hR2 HER and HER1) subsequent downstm patway molecules(fg, P13K) and non>ErbB RItKs (e.g. IiN and erMEW has been developed. 1 The selected target is captured by target-pecilk antibodies printed in serial dilutions on a 2.5 microarray surface, Thlis fomaat can utilize co-locaimoion of two additional determr-~ antibodiAs linked with enzymes (how n Fig. 24) 2, The immuno-compiex formad by the initial target bing by capture antibodies and the secondary binding of Glucose Oxidase (G) conjugated antibodies that recognize alternate epitope on the captured target molecules can produce HI02 in the presence of' 30 the GO0 substrate, glucose 3. The target specific local infhu ofHO2 is then uilized by phospho-peptidetpecibe antibodies conjugated with hornradish peroxides (ElR) that bind to the captured target, Specliicity fo the detection of phosphaorylated targets is greatly ncre ased tvhrog the requremenlt fox rouitmeous binding af three different atibodies The detection n and' rociel cidv d hhi quamificanon of as few as ~:24 x 1I Y phosphorylation events is rotnl4ciee yti method, 5Sde Pnutnig: A non-ontact microanra printer (Gesim) wsvo Osed to print on 16 pad nitroceliiose FAST slides (Whatman). [0424) FIG. 2$ shcays the detection of total. and activated full tengt and truncated HE2,. BT474 eel iyste whIeb was immoprecptated with Ab to the extracehdar1 domain (EC) or the lintraeellar doram (1CD) of HER2 fo 'owedby western blot with Ab to ther phospho trosine or the ICD. 4251 PHI 26 sheos deatein a otal ad phosphoyhted p95 Cpltre atitdies to an ataelir vERI epitope (ECD) and itraelular HER22 epitopes QCDt C2and 1ICD3) were printed 1474 ccl ivysatc with 4e indicated numberof ces was tested and the pression of totandin ted p95R$ER2 in wasdeternin ersdts tdemontrmt a 10426] FI 27 shows tetNmentwith Herceptinncreases thieed of actvaion of full gand n eated HER2 i m"tant d. not senstive cWlls SensitiveBTY474) and resistant (1R1) ell were treated with Hderceptin fardaffernt tines and ivsed. OUPls s nal e fresozesi n d ando oyted11 (and (rY ta and phosphoryoea p954QER2 (DP 104271 FI 28 shows tnreanmen wh Hercet and leveof actition of:ER3andf P o sensitweBT474)andristant ( R) cel t differenttime oiod MARY 25 [M428 Ful length a nateud1"' H12E.T w : analyzed in a highly sense mudtiek ff4291 Herceptimresistaut 131474 cells showed significantly inghem gpho ryiaaio p95HER212 than. .erceptinsitircell upon treatmrent witherepti [0NYj tpression of t HE1R2 and p9 tER2 decre ased' bh sensitivad resisant 30 cae reatedwith Hiercepd 124 O431jHER3 and PI3K phosphorylation in ells vtrme with leercepd higher in res stan! elis at early time pains wile at later mits the levd o actiated ER3ad P3in s:ensitive and resistant cells are stiaru 432j i R and cMET expression and phosphorylal lOvals we c omuamble W, 5 s nitve and resistant 47 celt [04331 Due to its high specificity and sensitivity this assay can detect fll length and runcawd .ER2 in limited samples and has ihe potendal vo be unlized for circulatig rumor cels and tine needle aspirates. Analysis of pathway activation and Potential mchanis of resstnce such as expression of activatedt p95HER2 can he used 'to select targeted therapies 10 most likely to benet a particular parent. Amd yis of serially collected samples can provide insight into he level of effectiveness of targeted trcaflnemt and enable phtysicians to adast patient therapy over tme Exakmpe 10, Therapenute implications of detection of av-preian and activation of HER2 and other receptor tyrosine kinases (RTM) in circulating tmor cells (C(Ts) in 1$ rcrrent breast cancer. ABSTRA CT [04341 UER:2 is on of four transmembrane RTKs in te epIdemI growth. fctor receptor family, and HiER2positie phenoype has been associated with an aggressive subtype of breast cancer (BCA) with HER2 gene atnplitication. Appmximately i$-20% of breast 2(5 cancers are considered HER2-positive by TC or FISH1 ena lysia Resenmdy, changes in H1ER2 expression status between primnary tumor and. CTCs found in recurnt metastato disease have been reported to occur at a significant frequency, FuYnctional profiling of HER2 in serial collected CT> may provide valuable insight into the overall disease profde shit and therefore lead to better selection, of therapy for individual patients. Whole blood collected 25 Mrm 51 metastatic cancer patients in two cohorts at multiple time points during the course of therapy and 605 healthy volunteers were analyzed fort CTC-ilERk2 expression and activation, We observed significant HIER2 status conversion with. recurrent disease, Approximateiy 30% of patients with HER2 negative status in the primaty tumor showed HER2-overexpresson in isolated CZT~s, Phosphorylated ER2 receptors were found in 53 to 60% of patients with 30 printary HNR2 negative disease 125 ITrRODUCTION 0435I lnterrogating the primnavy rmot in order to determine potenia responsieest targaeted therap has bectome the standard of car'e, Fuh chtaternztion of target expression, vacation. and downtram toil signalig proteins is seldom performed however, Changes in 5 the pattten of R1<{K express i in tumor cell poptdations during the <ite framte from. initial diagnose to recurrnce of metatti disease is virtually never asessed. A good concornane between HEl2 gene statu in the prtiry tumor and in coresondmg CT~s was reorted When ampl>ere obtained syn hroncusly. However, CT s frm retpsed patients w nitat HAER2 native primary tor sowed that CTCs can acquire HE*R2 mpificaion (1) dmonstaing substantil discordance betwee primary and metastatic lesion of sufiient significnce to ber dsease management Signi :ficant discordance in HER2 over-expression between primary andl metastatic sites has been reported using 11HC in. breast cancer (2), and acquired HER2 ~t geneaphfictiont in CT~s was confirmed by another ru 1,Ti dease poie shift may be uto th: aeuifan ot pressures oto the 2 etsrOgetmta 15 Dwnr Qe!0 populadton ofmany cancers that cause pattems of ceii-signaling to evolve over METHDS, oi a u m MIs enzye;LW 2 46J \iirr d I5ximit ssz; i h Cilaboratv Ptoximity imnv OAay (CO PtA)i st based on ar multip1exe.d protein tmiercarray p 1 tform combined with a traP a0mibody-enizymie chaneog ign samplifiction process. The unique and novel design is prvie y:he tripl-mtibxxdy e.m aoppuoch that centers utrashigh sensitivity whl preserving sp'ci 4ity: (i1iThe selected target is captiaerd by taSPCetpecfc antibodies printed in. serial diluton n ad mioarray surface Tis format requires a co-localbzadon of two additonal detector-antibodies liked with enyes (shown in Flxi. .29)~ (2Yl'he immiunn 2 conplex frmed by the iritia target binding by capyure antibodies and the secondary biing of glucose oxidase (GOY) co:jgated anti bodies that recognize aiteraie epitope on. the captured target meulnes can produce 1r0d in the presence of the 00 suibstmate, glucoce, (3) The target speeitic local influx ofE H02 s then utilized by phosphopeptide-specific antibodies conjugated with horseradish peroxidas (HRP) that bind to the captured target fy o Ythe detection of pshosporyated argts is gratly inercased through the requirement tor simultaneos bindig of tree liEren antbodies The detection and quantificat of as tew as 2-3 x 10Q phot.sphot'yation events is routinely achierwd by~ this method bringing its detection to a tigeP cell levei. An exempl~ary slde .onfiguration is shown in FIG. 29, 2 ~66 10437]1 ctli ghor; Ptients with istologicay codirned solid cnoen t regional lymph node or distaannmetastase a ta 1 on 4) SubgCswith Stage breast eardinomalhadregion ytm.node staging of N1ON>2. r NI Saxnpkes were ciected regardless of their therapy sttn f P943jt 080nc02 Cohort: Patients with progressive, evduable me.tastti stage IV breast cancers and who are about to start systemaw therapy . ent of disease in both cohorts was deeminmed by physied examination ad imaging stdes as pers the prnmary phyician. The tests utilizced may include one or more of the fiolwing bone scans. PET/CT scans, CT of the abdomen, chest radiograph and/ or CT of t chest for visceraletastases sonogram 10 andlor MRI for sofT tissue disease, REST AS [O439i The referee ulvaes were estabishedas on o a saple 'The dtibutlon of activatedHER2 R2) ep2 ssedE2 (t RStad the l Ch sunteared in$1 30S 5 4401 o The p'values were calculated usiug a two-sided Wilcoxon rak sum test, a nouparamietric test aimAr to a -test, AR of the comparisons show a statistically significant difference (p <-0,01) To determine: pHERii2 ad PHERS .stus, the background was subarat~c ed the signal was weighted based on le vel of CK expression, The conversion n a p r~ at' ER2 satus in her T. s are summarized in Table. 6,ERS status conversion 20 among the 080jcO2 cohort during the S offollow-up is shown in FIG 3 1. Co-met and tmmet asesmn is shnov in~ Fnc 32. Tabl 6.HE2 statusroeson int CICs. CR0n:Oi Studp 7I($~ tk~nJ2S~y20\& 12 IGOV (0% 25 into Whcle bloodt were i ranunomagnste y al islted and analyzed for HERdstaavs Corresponding COPIA.-based ftungdonal HER2 protiiintgi s~unnnatiaed in FIG. 2 I{N21 fiet mp iu.rae a noed tehooiogy wih unparalleed sensmnt and 127 cancer patients. Analysis of CTCs frn metastatiQ beast cancer patients showed tat 60% or patients with HER2~negative primry tumors had CTCs with activated vHR2 white only :40% showed over-expression of HER12. 'The mnmer of paints with activated HER2 and overepression of HER2 iraed over 12 weeks of treatment, The fusnctional protding of 5 CTs b COPIA vs, FISH xas correlated, An assessment correlation between CYIA and FISH fo analysis of CTtX HER2 status hn BCA patients can be pefrmed. [N4431 The OPression/aetivation profiling of kinases and oher signal transduction pathway motccukes on a serial samuping csf CTCs can be performed using the COPIA platfrm, an this provides vdahie iCnoration on changes occuning in tumor cells as a fiction of time 10 and therapies. This method provides guidance, not only for initial selection targeted therapeutic, but also in subsequent monitoring for rapidly *evolving 5 cancer signatures in each patient. Our finding of ER2-converso may be due to clonal section of HER2 positive cells whini heterogeneous primary tumoer cell populations or gaining of gpfneti( capacity tor over-expression. (. gene amplficatioM) Regardless of the mechanism behind 1 HER2-conversion, the presence of 1-ER2 in CTCS requims clinical attention, REFER RNCES [0444J I.. Meng S, Tipathy D, Shete S, Ashfra R. Haley , Perkins S, Beitsch P, Khan A ulms D). Osbomne C Frekel 1, E oov er S, eitch M Cliford T Vitetta , MorrK on T, Herlyn D, Terstappen L. Flenning T , Tucke R, Lane INi W J r ,-.2 20 gene am5plfication can be 3equired -as breas cancer progresses, PNAS 101.938 2004, [0445j 2. Zidn.J, Dash*:cvsky I \tayerman C, Basher W, Cosacov C, iiadary A, Comparison of HER~&2 overex~pression in primarys breast cancer and mretastatic sites> and its effct on biological targeting therapy of metatai di\sea B-r I Cancer 93 :552- 2001 [04461 3 Hayes DF, Walker TMs $nh Bw Viteta ES, Uhr JW> 1oss 8, Rao C, Doyle 25 GV, TersaMppen LWK Monoring expression of HER~2 on cicudaing epithelial cells in patients with advanced breast cancer. Int Oncol 21 1114 2002, Eampic 10 Prevalence of acivated & total p95lER2 anid other retcptor tyrosine knases h breast emncer, ABSTRACT 320 104471 HERP overcxprcssing beast cancer B0CAlhas poor prognos s and is often reitant to -HER2targeted mnn ona antibody therapy. One the ewchanisn of de nc- or 128 acquired resistane is expression of vatIous3 forms aof truncated RR receptors with missing aminso-enninal extra cellular domains, collectively preferred to as p95HER2, Mdetho for prmiing various forms of H1ER2 receptors and other receptor mymsiae kinases (WEKsi with trasacttvation potenta" in primary' an m t astaric umors may provide valuable the shi ing disease pathogenesis. This cxmple describes the successl prodigy of a panel of signal rasdion pathway proteins for t rpression and actvation in i10 frozen primary breast cancer tissues and A FNA samples collected from tetasatic sites in breast cancer patients wih various I1R/PR/(ER2 stats, INTRODUCTION 1 0 [M48) Several mechanisms for Trastuzumab resistance have beenre td Primarily, the activattion of other RTKs (such as iF1-R) and the accumulation of truncated forms of HER2 have been frequently repeated, among other mehanisns, ia paricular, the ainoennind trnmeated carboxyl terminal fragments of 11ER2, collectively known as p95HE/R2, are frequently found in HER2-expressing breast cancer cell lies and tumors. CrOs between 5 various sna transductionpathwas a d pS provew' e ecaim for tuors under certain therapeutic pressure or pathway addiction requires a comaprehensive diagnosuicn et pro p network nasis. Treatmem decis.ons made based on clinical infrmNation obtained through current IH C/FiSH based techoogy pcrfonmed for a few selected biomatrkers will nt be effective in treating patiensts with tapidl e .volving 20 hetemogeneous disease, Ftuttheore, existing technologies not only are limited as they can only provide 'static andc limited' information, but also require substantial amount ofhtises. Obtaiig sufficient amount of samplescoulid be quitcalenging, and reakime disease monitoring is nearly impossible, The unique asay ptform described heroin prorides exttremne analytical specificity, aliowving rmTiplex analysi with a limited sample amount. 25Differeam configuzradion of detector antibodie allow differemtial detention of truncated targets (e~g p95HEilR2) fromt their fli'ength coume rparts (e,ge, H'ER2l. ins this study, the faustional status (exp'ression ad activation) oflHlER2, p95HER2,~. HElR1, HElRLK riadi IGF as we'li as the downstream signal transduction p~roteins PD3K, She, anrd c~METI were analyzed, M0049~ Mutipieed Proitinst Asay: The C~llahorative Proximity Imumun Assay (COP IA) based on a tuniplexed prden. micraayplatfdrascombinedvith trile antibody-enzymaechanneling signal ampliiation: process. he unique and novel design is provided by the triplh~anti body enzyme approach that confeibsltrahigh sentiivgigty hi 29 preserving specificity: () ~The sekoted taget is captured by targetspecific ttibodies pri tedin sria diutios o a irva surtace. IThis format requires a co-ocalizati of prned ise~).faiot nne &Y4 idand two additional detector-antibodies linkced with eryxymes (shown int FIG. 29), (2) The imuna-om~plex formed by the initial target binding by capture antibodies and the .5 secondary binding of glucose oxidase (G) coniagated antibodies that reonz ttenWte epitope on the capt target molecules can produce HQ02 in the p e oh sstrate. glucose, (3) The target specific local influa of' H2O2 is then utilized by~ phospho peptide->speific antibodies conjugated with horsc radish peroxidase (.HR5P) that blad to the captured target 10 dse~ Spe"ifity fir the detection f phosphorylated targets is greatyincreased through the requttmmnt for inmtaneous binding of three diffent antibdies. e detection and quanilfsatianof &as few as N- x i0 phosphorylain events i Uey achieved byhi mt~dd bringing its detecun to a"singe" cel leve [P451 Frozen issues; Frozet breast cancer issues were ftrn am asian patdents with 5 duca breast carcinoma at stage Ii oH Ii. olleced tase samples 'were lysed and stored at 8(YC until theperfbrmance of the proxity assay. SaFies ~A ssnn~e yr" 0431 QnigdMga N Asamle wereA~t collected from patients with progressiv measurable metastatic Stage tUB, anid/or Stage IV breast cancerad who were about ws start systemic therapy, Parents mu st havc histologically or cytologicafly contfmned invasive 20 breast cancer with ECOG pcrformane status a-2 (Note: ECOG 3= Capable of n ii sef-care confned to bed or chair more than 5% of waking hours. All patients had dist metastasIs sites of disease anmable to biopsy. The FNA samples were mllected using 023 gauge needles, and were analyzeo expression and tivation status for various RTKs and downstream sigsnal tramsduction molecules including p95HBR2, 511R2, 11PR . HER3, iGF-~ 2Sc; 4Rw, P and Sa. FNA rampis were processed immediatey o sie of collection with "Proteintaterh ell lysis budior before shipping, RESIUNTS 104531 P r x in 10 imar B se Yhe I r osi d' p: R'N t N BCAtssues Wth different HER 2 expreio stas is shown in 4(A) The lev f 90 HER2 phosphorylat ionin sampcs with varying ld ofp9 h is shown in FI 0. he clorof the dot repTeets the 14ER2 status determined by ,,i 130 [454] lopiioqes cgi G TtA; FLG. 35 shows the correlaion beween the HER2 depression status determined by H or/and FISH vs, COPTA, There was approximately 15% discordance between the twc methods. .iP-Westemn analysis was perfoned to confirm the HER2 expression states in discordant samples as shown in FIG.36. The H1ER2 status determined by COPLA showed 100% correlation with P-Westem analysis, The ighlevls f dicreatiy btwen HER2 status detenrmnd by IHC/FISH- have been The h ih vealsA of discranic beu , _v__ reported by many groups previously possibly due to procedurainterpretational variations or tumohtrgenety. 1455, k t gygts; In additionto HERAp95HR2 and OKhe level and express on and the degreeaof activation of other pathway prumits we goalyred for BERL THR IGFi P cML e-KEP 1 U nd Sha f04561 Figure 37 provides enampies of functional pathway profiling by COPIA, in. case i, both hER2 and R are highly expressed, hA only E2 i a e Case 2 has ve high level of HER 1. and some significant level of HER2 and HFR3, but does not show any S meaningful activation, Case 3 shows some expression of Hir2, when the RER2 signal is compared to theC signal i clearly shows that TIER2 is not over-expressed (or amplified) in this patient The HERZ expression in case 4 is a good representation of HR2 vr expression as the ratio between HER2 andI CK is s-gnifiantly higher than case :1, 10457 The levels of activation seen in frozen tisue samples may ot represent the bas 20 line in v unctioal profle due to variations Iat e processing post-surgery. mmed iate processing of coLected samples is desired to n tor m dro tmotional profile of pathway proteins, "Proteintater" is suitable for immediate sample processing post-'NA procedrtie. [0458] ENA Anaiysis: FNA samples were collected fhrtn metastatie sites olBCA patients isolated cells were immediately iysed in "Proteinhate and were shipped Wbr 25 subsequent functional path way pruling, One aim of this study was to identify patients who would respond to targeted inhwbitors. Another aim was to idetify patients who would bxnet firom a combination of targeted agents, FNA samples were collected prior to treatment or during a I week therapy holiday. 046 The eailuissentacts obtained horn the FNA same we aaysed for expression )0 stivtion at us of hefloing signal mansducers:EB RI. Erbf2LER2 I3/HER3~ eaet, IGFAR. Kit 113K, and She lgG and OK were ud a con 1G. 38 shows the miroarray slidformat used. o this study Total andhosphyated 131 p95ITER2 keels were detected on a different slie, Addtona signal transducers ftor which the activadion and/or expresion sttus can be detected include, but are not limited to, Aid (Ser 473), P70 S6FK T229), Frk2T202Y204', RSK (l59/S363> Stat3 (Y705) aw combinations thereoE in some e iments, FNA samples can bend ten 5 concentrations to provide qtitative expression level and the degree of activation, 1O460 Functional pathway prflig on FNA samples using the COPIA piatibnn described herein is particularly advent ageous because it is a quantitative method in which cell number. recept epresi" nd receptor activation can be obtained with a high degree of accuracy, FIiS. 39-46 provide examples of pathway profiling on FA samples obtained from patients 10 with metastatic breast cancer. The table in each figure provides quanitation of the total and phosphnrylated levels: f each marker in relative light units (RLU) Table 7 below provides a srumary of the e>presson and activation staws of particular signal transducers detected in. thee FAampeS and also provides a recommended course oflreatment, *02 o~nt erah( e expressed .~Ft~i II wift'p 1 8C 00l03 Wea patwn cpression N . Nedh tj kio htonxnai satus low IwoWIN ttZ00S%0O4 HER 2. IE-I f R, eMet NITR xad fGA ftR Acsti-lGP-R therapy exprsadactt( apattab + hnnomi therpy) 5C3 05,065 HER > 1GF-' eIes H R2. HJ'4 fd uF R Treat wvtn )apauib todeily %t<pr"sed acitd 8034004006 1 t 'a RIR, lct&et 1HER2 atod HPR.I hype& 'P'ret wtt *panih or arni modi~eriely t:<pr4d" ativated (H10272'" 8C34007 ,HfR 26vm-expres: iR2U StHIactl{Cted T ns Woh acipna L'SchemO seVOil5.608 ER 21, C e eAt None Need to know ItormouIs stit' rnodertely e&NpresaSd [Q461] Multiplexed COPIA pathway profiintg performed on.primnary frozen tissues obtained from BOA patients and FNA collected from tetasic tes siehowed: .132 OC55 % concordanee between primary HER2 tIC ~wu nd CQPAdHR2 exprssie ~?eeneo sigyificant p95.HERP in ove 0%} of H2R2-posivc HIER2s 3 and w±;ith ISITS)p aents and detetabe leves n soe tissues with other Nstoogy HER24HC 5 Wth02 WISH A 11 A ~25% of RER2 possyc samples also had other KI pressiorn and/or activatia 44 Thes resus show.heaerogeneiv iR1 expression aid sgnaing paahwar 1 theapie tnmp 12 ihnettaaiprofding at multiple signal pathway proteins in beast maee patients jO4634 The C~ilabonuive Proximity onnmnAssay (COPlA) is a mutipiexe protein microarray platform that nmilizes the tbrnation of a uique hatn oomlex requring ena $ lcalizat on of two detector-anmtibodios. The detector-amibodias are conjugated wath responding chbannelng-enzymres, glucose oxidase. (GO) and~ hOrmerudibhpseoxidase (H RP) Once target proteins are bountd by the capture antibodies. the channelling events between GO and HRP in pnrximity enables the proving oft t target proteins with extreme sensitdvirv. COPTA delivers extremely high analytical specificity as it requires munipic 20 entities within target speciftg proximity for the signal genermiov/max iication COPA can also be conigured tor each species target protein to ahow dibrenial detection of truncated targets (e g. p95 HFR E R2frm their normal couterparts (g. fuliength HE R2). COPIA was applied to investigate dhe levels of expression arnd acti von of HER, H.ER2, P95HER2. HER3 (FlR M

T

, P3K, She. VEMF, panCK and other targets in sigal trsduction 25 pathways (0464) This example daenstras the functional pathway signatures for multiple proteins in 250 zen tissues obtained forn BCA pants with various priary histology and fronm $0 ine needle aspirate (NA) sanpes collected rm metastati sites (mFNA) in advanced BCA patents with varios ER.PR-M 1R2 status. There was a high conordance between pnmary 30 HER2-iHC status and COPEIA R2 expression analysis $igninmt levels of p1ER2 were observed in over 47% of HE3R2-positive Zp s ER2: 3+ Id 2+ with FISH) patients and 133 low ut detectable level in some ape tissues with UHC-HER2 negative (2+ with F I + 0) were adso observed. Over 50% of p951iERl2~xpressons had. activte p95RER2, and over 25% of.HER2-positive sample also had ER1, HER TGF1-R and other RTKstad ransduction protein pression and/or activation, As the disease profile often thins int S recarent breast cancer, tho unique assay format described herein can be uirlized to provide valuable clinical information on limited samples obtained from evolving disease to help oncologists adjust their disease treatment options for each patient accodng to their personaP cancer profide-shft Having the ability to profile tumors at difrent metastatic stes with an expanded pathway panel provides information on their diferent:al metta 10 potentis; hence mnimally invasive single-passag-mFhNA samples can be utilized to tailor therapy options accordingly. Example 13 Charaeteratbirn tf HER2 Fu-ciona Profiling in Fine Needle Aspirates (FNA) in Patents with Metastatic Breast Cancer (MBC). 0465 .Tavproguld The C laborative Proximity immunoasay (COPIA) is a muliplexed U micoarray pIam that usiloizes the formation of a uique imtoompe resiuirin co localization ot two detector-antibodites. The detectorantibodies are corpugated with corresponding rhasmelingenzymes fOr proximity mediated signal generationmpliticaiion for the expression and. activation profiling of HER!, HER2, p9SHER2, HER3, GFt-R, v MET P13K, She, VEGFR, CK, and other signal transduction proteins. This example shows 20 HER2 expression and activation profilrng in tine needle aspirate (N NA) biopsy smupes frm MBCO patients with this novel technology. 0466) Mvthda FN A samples 'were collected rom inasttie sites from female patients with poaressive, Stage IMV breast cancer (N=5. baseline age 4213gr)e Pats with various clinical primary ER PRR/HER2 statuses by IRC staining were allowed in the stud>

X

5 The ER!P/R ER statuc u he primary m apl wasoi b t oc Expression and actition status tor HER2 and the other signal transduction proteas (STIP) were measured nung COPIA on FNIA ramp lea 104671 Reg Data from the FA samples of' the first 10 patients showed that 5 (50%) were HERS activated (pHER2.+L 2 (20%) were HER2 over-expressed (tHER24), pHER2+ 30 and pWHR2 over-expresse, '7 of 10 ENA samples expressed weak to modeaste levels of HER.2 wih different pHER2 levels. HER2 status (expressoon/activation) was negative in 1 p3~atient. The concordance between the COPlIA-derived data from rthe ENA and the primary 134 tumt IUC wilhe evaluated n adding evrtxpsanandko ac to of other STP inehidWing HERI, ER3,F RI It and e4E wi heneasered in theetir cohort using 104681 Casio o MA assay can be used to cuntita&epression ad artia&n of S', j NA speimens. These results find value to infarm keatment deiionsnrelapsed breast canoes panerts. Example 14. Alteraton~ tf HER2 Futnctiotna Prof1iig Over Time in Patients with Metastatie Breast Cancer (MC), 10}469J Makgang4Q Blreast cancer patients with HiER2 over-epresionapliication~ are 10 assorted with shorter time to relapse and. shorter diseaseee aid overall survival. 15-20% of hreawt cancers are HER2-positive by IHC ox lSH, Chnges in HER2 status between primary and metastatic twdrs have been reported to occur and assessin this might have theapcude impact, This example shows the convesion of i1ER2 status over time in MBCZ patients in serially collected circulating tumor cells (CTCs),. 1 5 [O47tA Methods: 50 fmale patients with stage iIIB4V MRC (baseline age 57-13yr) of various primary ER/PR/H1ER2 status, determined by l!HC stainng were enrolled and followed up to 14 w eeks where they received various MIBC therapies per their physician's discretion, Whole blood samples were collected at three study visits, 57 weeks apart from each other, for isolation of CT~S. The seau CTCs were counted using the Veridxm od 20 and rested for eNpression and phocphoryiation (activation) of UERi FER2, and CK. usin a novel muldtiplexed microarray (Cofaborative Proximity limunoassa, ChA) Tis platform. has been developed to enable the profiling of the target proteins in signal transduction pathways at high levels of sensitivity and specificity Radiographic turcox assessments were condted at the 2' an- 4vii 2Y (04711 es; In primary IHC-HR2 negate brast ntnos 30% were HR2 over expessed (THER24) and 56% were HER2 activated (pHER2+) in CC beikoe initiatiw a new therapy, CT0s 5 were observed in 37% of patients and. CK levels correlated with cell counts, Patients with no detWcl T~s or CTCs <5 had unpredictable levels of CK, however HER2 expressio or active ation. was detected in CT0's inl 31% and 47% of those 30 patients, respective ly, A7 of the patients with CTs S5 had an. 11H C-HER(2 negative primary tumor 11 out of 15 patients (73%) were ER/PR positiv' 13% of the patients with CT Cs 5 had. IUC~iER2 positive primary tumors, all were ER/P neaie 6 d i 1 f07 C ngsos R2 status can be measured ini CTCS with C2)Pta: heraton of RR fanctioad profding oer timte in atients with MIK has been observed. As such. thi example lustrates e v e of CTCs as a potent source ofti fn v prognosisand diano sis prtitng. E1mnpe 15, Analysis of Trneated E Expression and Attivation in Breat Cancer W4 7 3] flackground: HER2'overxprssing breast cancer has a poor ptogse andis often resistant to 1hR2 targeted muonoclonal antibody therapy. One of the me chanisms of de novo or acquired resistance is expression of p95HER2 wich is rcini ay associated wi aggressive disease, poor prognosis and lack of response to Hcrceptin, Clinical studies on 0 p9511E3R2 are liied dtoe to lack of highly senave and specitc assays to aceurtety measure its expresion and activation, 1U474]~ Methods: A4 novel tehnloy cbl of specially detecting expression and phosphorylation of receptor tose kiOss (RT)(CmA oa wsused tO detect op5HER2 a full length HER2. This multiplexed proein rnicxoarmy platform 1 requires the co-localizatioa of two detector enzymeonjugated-antibodies when in proximity enablec the i o the" RTK wit ig sensitivity. Using this assay, weN analyed the expression and actvatin of 5HER2 as wel as ouher major oncogenic pathways incuwdng MR. HER2, 1H1R3, 1GF A. -MET, P13K. She, VEGFPR, and CAI in 229 tozen breast cacQrC tissues (stage . to 1), Expression and activation of HERZ, p95HER2, and P13K 20 were also measured in Hlerepiin resistant and sensitive BT474 eeils, [04,75 Resnlts: This example describes the successMI profdig of HER2 and p 9 5

ER

12 expression and activation in frozen primary bast cancer tissue s adetrmoed by IC and tetd in COtPIA4 Approately 39% of 11HC 3+ samples had activated p95HER2. The MA,, 1+ as leW of expression and actvation of p95HER2 in the UH0 2, twand negative subsets was 25 damaRically lower althoNgh with some tissues remaining igm ca-nt All 10 markers tested revealed diverse activation of signal pathways and heterogeneity, in our precinical studies, Herceptn-resistant WrT474 cells showed significantly higher atctivat of p95HIh Mll length 1HR2 and P031K compared to Hecepinn-sensitive BT474 cells upon Verceptin treatment. Increased activation of HER3 was observed during the first .24 hour of Herceptin 30 teatnen 194761 Condusi The highly sensitive and specific p H COFIA assay allows accurate detection of fl4ength E2 iand tota and activatd p95 3 in nv tumor 136 sampltes such as fin needle aspirates or co re biopsies Quantitation of p95HER2Z activaton could select patients who ae most likely to respond to Herceptin The COPIA assay enables detection of ultiple kintases simultaneously in the s sampkes which sends ilit 3n the mechaism of p95 IER2-associaed H p resistance , p95HEP2 expession and 5 activation can be measured in clinical responders and non-responderstHeepitrane. As sue. analysis of changes in p95HER2 expression and acwarion over time, whether treatmemnrelated or due to the natural course of disease, enables more effetive selection and adjustment of therapy for inrvidual patients, Example 14. Expression and Activaton Profiig of' Receptor Tyrosiae Kiinases Through Cyaborative jProximity Imnunasaay (COPIA [0477 This example provides a further description of the formation of unique immun comrplexes on an. antibody-amieroarray phltform,. In ome embhodimient on~e of the detector antibodies is cojuigated to glucose oxidase (GO), and the other is conjuated to hotseradish peroxis (H Th aRsy speificity and sensitivitys enhanced given that snal is 1$ generated when imumuno-comaplexes are successtfuly formed and amplfied through thbe enOzymC channeling between coocasized ( and HP>on captured target proteins, This method can be applied to profile expression and phoshorylation of relevant biomarkers in cancer samples, The methods are useful for the predictionoaf potential treatment responses which then leads to better initial selection and subsequent montoring of targeted thrpies, 20 IO4781 The immumoassay described herein provides functional profiling of signal and the bili onitr teproie shift in cancer cells. The methods provide valuable insight into oratl disease pathogenesi. The methods specifically detect phosphoryiation events in Erbl family receptor tyrosine kinases (RTKs) at a single cell leve INTRODUCTION 5 104791 Multi-target assessments of gene expression in normal and abnormal tissues have expanded the understanding of the0 pathophysiology of many diseases. While nmRNiA profiling eant provide valuable biological infoormation, its el inical potential may be limited due to multiple causes for post-transcription defcts. Despite these imitaions, advances mnade in basic and translational research have resulted in the incorporation of semonntes 30 technologies imo clinical use for complex diseases such as caner, thus paving the vay for new genomie-based patient management (Pailn er at, . Engf I Med 351, 2819~26 (2004); Paik S. et a. ; Cin. (co 24 3726-3734 (2006). Multiplexed genomic-analysis matured due to the exqoistte senanivity and specificty tf olecular technologies based en steuece-pecific target amnplificaion processes, in. contrast, protonmic-based methods have ot yet developed into a praeticai emulipiexed forat Most current proteinbased aplicationas ae based on trditio~nal imtmohnistochemist (JRC) principles and. require a 5 subsanial amount of sampe. The ore successful cinical application of protomics technologies avwaits better sensitivity and speciticity, More importantly, as the activating (or phosphorylation) state of' the proteins reflects their impact on cellular fimcios1, a proteomuic diagnostic plat form must be able to differentiate the level of protein exprressiont and the degree Cf protein activation. [0 f480) One of the most widely used applications of proteomaic assessments to therapeutic atnd prAoostc outcome has been with the detection Umfhun epidermal gowthtot ect 2 E pressnn i breast canc (BCA) patients usi 1H4 However, this method has teheMicl litanions wit analyical sensitivity> target specificity, capacity to multiplex, and subjectivity in image interpretation (Gown, AJvL, Md, Patho. 21 ,S-815 (2008); Rhodes A. et , CiJ Paho0 53, 125-130 (2000)). urtthennore, significant levels of discordance between the results of HER2 studies performed in diffe rnt laboratories have bee repored (leddy, AC. et at, Clir Breast Cancer 7, 153-7 (2006)) Hence fluorescence In Sie Hybridization (FIsH) technology is ecurren used to detect HE R2 gene amplification when the JUGCbased results are ambiguous. A staged use o bth technologies 20 is used to determine patient eligiiity for trastuznmab, HEWR2 targoeed therapy for BCA parents (Cadirs, M. and Villegas, IM. Apt t mnm Mot Mr I7, .7 (2009)) A further imitation of current assay methods is their inabiliy to determine the activation atus of the target protein. 104811 Between 20% and 25% of invasive CA patients exhi bi ower-expressed 10ER 25 Rr T'Ks (Sannon, DJ. s at ene 4 n 23$ 177,82 (1987)). Over-expresion of HER2 r .c k proliferation and disease progressiom and HER2-positive BCA has a higher recurrence rate and reduced survival (Son, Dl et a, Science. 23.. 77~82 (1987)), Determining the HER2 stts in BCA patients is critical as its status is integral for therapy selection (Cuadros, M4 and Viliegas, M. Appt Immun Mot Morph 17, 1-7 (2009); Siamon, DI et at, 0 235 17 (1987, owe vr , ony approximately 50% of HER2-positve pattens initially respond to trastruzumab-comapemented treatments and subsets of these patients show inherent resistance after having a dramatic initial response or will eventually develop resst ance (.Nahta, Rh and Esteva, FL, Breast Cancer Res. 8, 215$27 (2006)) Although HUafliKC and 138 tER2-FSH are valuable for prelHminary patient selection, neither test can differentiate .responding and norspordig patients. Therefore, there is an urgent need for the development of reliable methods io differentiate whichi HER2-positive patients will respond StoHER2-argetinrg agents. Such tests should ibe able to determine the Pmetioal state of th e SHER2 protein along 'with prfile of its potential heterodimetlan p eioder to provide vital infonalton n ritional selection of the most effective therapy option, {0482) As tomo are exttmely hetereous, the cells in the primary site may not elect the: profile of the tumor cells in recurrent disease. The more relevant soarcms of trunor cells for guiding therapy might be the metastases of recurrent disease, An alternative soure ot tumor cells is the blood where small u-opulatiors of tuonrr cells are foIund n fpgressiv disease (Cristoltuilli, M. et at, K .EngL I Med, 351, 781-91 (2004); Hayes,. DAE eta, Cla Cancer Re; 1-2, 4218-4224 (2006); Pachmann, K, e at, Clin Oncot 28, 120> 1215 (2008)). The tunbher oft tumor cells in blood depends on the stage and type of the teuor and varies fromt undetectable to several thousand cells per milliliter oftboodi 15 (Crisaoi t. r atA1 EngL . Med 351, 781-91 (2004); Hayes, DE et at Cli Cancer Res 12, 4218~4224 (2006); Pa1hann, K. e ,! ClMa Oncot 28. i28- 1215 (2008); Nagrath, S. a at, Nature 450, 1235-14239 (2007 [043 CirculAbing Tumor cells (CTCs) provide an opportmAy to perform a rmin-ivasive 20 ^'eliebns"on mnetastatic cancer ptins. using the C~lliaborative Proximity immnoAssay (COPIA, Figure 24), which has uAirkhi senstivity and speciWt, and can detect the activation State otmultqple i transduction proteins at the single el! evel with an analyital senstNivity of about 100 zeptomoles (r between 1 ' it to 1 x !0& target monlecles), As described. heroir, COPIA can be used to quanttte the expression and 25 phosphrylation of HJE R and HER2 in various cancer cell lines, xenografts, 'rOZen tumor ssues, and CTCs isolated from BCA pate {484t Tming to FlG. 24 the individai components of target specific complex forming antibodies are shown, which are used to detect the activated ate of target proteins, The capture and detection antibodies are selected to mnimize competit ion between them (e, all 30 arlbodiws can simultaneously bind their corirepondg epitope on signal transduction molecules), The first degree of specificity is accomptlished by the interac tion between target molecules and their corresponding capture antibodies printed in for. exampleg erial dilutions. $19 .'k Meo anfchaeeliru The actiation tl-independent deetenantioodies are conj ugated via a -haadin tnetswy e glucose asxidase (G) and Ohe acthom state-dependent detector antibodies hbaed with a signal amnphfcation raietye~g. horseradish peridase (UK?) [0485) FIG. 47(a shows the activaton of 9R and HER2 at a sensitivity ievel of a single 5 cell in MDA -MB468 and SKr3, specively, These cell lines express approximately ito 2. x 10C HERl or liER2 RT is on their celil membrane per cellt Mictcriny slide ima~ge\s for 3, i, 0,3 cels and nengaive control are shown above the ce titration curv. MDA4-B468 ce-It were treated with EGF to phosphorybate HERI RTKs, while HER2 RTKS are qpmtaneously phosphorylated in SKIR3 ceLs. The cli aount on each pad was generated by serial 10 dilution. Capture antibodies were printed with 500 pl per spo in triplicates in sesial d'ibions of L7 mg/mL 05S mg/m .5 mg/n and 0,125 m g/m (I486] The westem blots shown in FI, 47(b) were generated from 12 gg of total otein per lane (approximately 4000 cells The level of dominant RTK expression 1 each ell line vas determined before and afte hE or HRO etimuilation. Thie degree of phosphorylatiern of 15 HERI or IHR2 was detected using phospho-tyrosrne specific antibodies, The difference between before and after uln for RER I ini MDA-MB468 provides information on degree of phosphorlation through growth factor stimuladen. [10487?1 As shown: in FIG. 47(c), the ranuer of cells required to detect 20% signal sturaton (or 2000 RRl) for pH ERI Dphospbozylatd H ERi) or ptl-ER2 (phphorylatd HER 2 was 20 used to calculate per-cell RPTK activation for cells with low RTK expression (RFcc) Non-deteetable signals in each cell lines were indicated as ND in he table. White pHER2 was detectable, .MDAMB 468 cells have -9935 RU/cell level of pHERI when stimulhued with EOF, Althoughi T47D cels express substantially lower levels oftHER I and :HR2 per cell, a significant level of RTK phosphorylaton was detected when t0 cells were 25 analyzed and there were dik'rentiail activation patterns when these cells were stinulated with either EFf17 or HRG110. [$0488 As shown in FIl- 47(d), xenografts we-re derived fromn cell lines with varying degree of ErbB-RTK expression: MDA-Mi-23I, MDLAMB435 and 3T474 (tmai 4 Y. er aL Cnce Rens. 42. 434~4398; Filnusa L et at.o A CelI Bio: 7, 251- (1987); Uherek, C, et 30 at, Bloo 100, 126573 (2002)x fragnwska. W-1 e at Mol Cancer Res, 2, 606%19 (2004T), We detected low levels of pHER~ and pHERQ-2 in M 13-231 xenograt, high level of pitlER-2 and signitcant level of HERi&l (due to co-expression with amplidthR~ 140 2) iFA sales otained from a T474 xa ry 1ow R-1 a RER-2 activation were detected in FNA obtaned frome MD)A-Ml~435 xenograft. 10489) A show in TI 47e, tissue samples from6 agelS to 111 frozen BCA (12 of

HER

4 .HC 3 7ofihER24 +, 7 ofHER2-IC ) and 4 normal adjacnt tissues were "'analye for HER2/HER expression an activaron. All prinary tumor samples with high RIC score (3+) had high levels of HERA expression the asay, and showed a high degre of aedvation of HER~2. The press ion and activalirm af'ER.reior detected-yor sa is concordanit with the tumor 111C score, For total RTK aalysis, RFUJ values weregerad fmom 100 ng of total protein, For phosphoRTK. analysis, RtFU values were generated from 500 ng oi'totad protein. B'4-74 cells were used as positive control for HER2 oxpression and acti ntian 0#49)j FIG. 47(f) shows a scatter plot of26 BCA saumpies for total HE1bR2 expesion and HER2 phospborylation, All 4 normal adjacent tumor samples showed no HiBERI expression, 10491] Tuning to FI 48(), fOr each slide, a standard cure from a serially dutied cell $ lysate was prepared from states of cell lines MD-468 (ER S-ositive) an SKlr3 (HER2 positive) cells. Each slide was configued to Obtain quanitative inforton to accurately determine the level oflTHERI and/or HER 2 expression and the degree oft phosphorylation. A total of7 pads (pad l, 2. 3 4, 11, 3 and 14 in serial dilutions) were used for standard curve generation, Each slide had a buffer control pad along with two quaty controls (pad 5 and 6) 20 Samlswre aad an 4 pad a 7, ,9, 10) in each slide, JO4921 n FIG 48(b) the limi of detection (LOD) value was detanhw d to be less than 1 CU for both pHERI and pHSER2 Near I cell sensitivity was observed for tHER2 and tCK. The LOD for each analnyte is calculated as the mecan concentration of the bufib control value + 2 standard deviations, FIG 48(c) shows a total of 27 breast cancr snampes analyzed for 25 their HER? pesso. and acti-tion are shown in the tame, 17 blood samples were obtained from metasisatic breast cancer patients with primary HER2 4HC negative status, Up to 59% (10117) of initial HER2IC negative samples showed evidence of HER2 activation in their CTs (shaded). CTWs with HER2 expression were found in 7 (shaded) out of 17 (41%) blood samples obtained ronn inmetastatic breast cancer patients with primary HER2 30 negative status, Levels of HER2 expression and phosphoryiation are shown in CU unit, 141 prednia ede r rmance fI )IA [1493j WeO u t iied tIssue eultue cell lines with Lnwn levels of I R and HER2 RTK expeinh to demonstrate the feasibility of COPIA on a microAwray forat MDA- MB468 eels were used to analyze the lvel of HERI expression and degree of HER tativation aost Epithemal (Growt Factor (EWF) Stimuatin a SKBR3 cells were used to detect the level of HER2 expression and attvation via protein ver expression. W.T474 and T47D Tella express sing varying levels of RER and JER2 as well as other &B1 RTK faly members were used tv show analytical specificity of owr assay method, Mavdieal Sasitvit 10 %t94j We de d he actvation andexpreson of TERI1 and RoR at a sensitivity level n A l ce in - MDA-M2D468 and SKBCR3, repectiy (FlG 47a$ These cell ines have ben~S wet-chVare for their ErbB3 Wl'K expresion by others (U.hcrek, C, et asBod 100, 265-27 (2002; trawsk, Wi et atl Mdi,. ancer Ret 2, 606~69 (2004) Harari f. and Yrden Y, Oncogene 19, 6104 V14 (2000) .Riethdot S. at Chr CeY er Res. i13, 920-28 (2007) These EelS lines express approximately I to 2 x 10 HER E or H4ER2 RTKs on their cell memat lowve3or, onl subsets ofdte expre se RTKs are phosphorylated when heated with gth5 factors (HER.) or over-expressed and sue phosphorya'tion level is adequate and sufficiem for the do"w stra pathway activation (Dragcwa, WV1 Ate at. MolP Cancer Reas, 2, 606-619 (2004)), Whhile 1RER2 ive ?0 expressing SKIR cells have Ronstitmive HE2 activwWat M D-MB-468 cells 'et in a nh sh state and need to be situated with GF o induce HER Iophorylation (Dt Sagowska- W ea al Ma Cancer Res. 2, 606-619 (2004); Ci tofanii M, eal>Cin. Breast Cancar 7,471,89 (2007 i). The dfferenil activation of RERl and HIER2 mediated by eitherEO (dijrect HEIRI stimulauon via 1hm 0r hterudimerifzation)l or 25 legulin (HiRG, indirect t stamuto vva heteraimerizationiwith HE3) mesnlts in cell lines essn vying levelIs of Erb fi *, K expression as shown in FG. 47b, While MDA4B-468 cells showed m inr HERt acivtion before situation, they show HER phosphoryiation upon EOF binding (FICs, 47a ad 47b) Typiaiy only 2-10% of bghly expressed RTKs are activated (approximatjy 2 a 10 to I x 10 ph osphoryiation events pet 3a .MDA~M,468 or SKEI cell) and this is sufficient fsr cell proliferation (Dragowska WL <a Mol Coan Re. 2 6 , The preset assay format enbd us to dee approximately 104 activation events, thus proucing single cel sensitivity (FTA 47a), 142 abjrah l Speifcith [0495] Thne anltia specificity of this COP IA .forat was extremely' bigh as it requires binding evets~ of 2detect antbodies to each target protein in addition to tha captrr antbdy. BaI, sed on a comprative study performed on muvap cell in with various RTK evels, t.h anatia specificty of this assay fonat was found to be geaer tha 9999 (FIG, 47). Whe N 46$ mells expressing extreey low amount o ER2 wereuse having ~.000 ceils per assay wa.s not suffti eet to detect (ND) any signal on HE3R2 capture sites, Whie pHE11R2 w as undetectable, MDA NMB 468 cells have ~992,3 RF' tcell level of' pH ERi when estimated with E, Although 7D cells express subtnti lower level of 1 H0 1ER1 and HE R2 per cell a sgnifiant tevel of RTK phosphot yon was detectd when I 02 cells were analyzd, and there were differential activation ptterns 'when these cels 'werec stimuilated with either EOP or HRG. Ast T47D3 cells express sinilicantly higher level of ER3 than HER1, higher HER12 activaton was observed 'when cels were activate with _RG vi HER2- HER13 heterodimerion formnadon> IHRG tmatmnent did not induce HE1 12 activation in this cell population demnstrating assay specificity On the other hand, E0F treatment of T47D els rested in both HER! and HER2 actvation though HER!HER2 bterdmeriawicon although at a Iower level than HR;J edia ed actvati on, Uetectable pHE11R2 in MDA-MB,468 cll iysat and a substantialy lower level of pin ECOF treated Str3 ells (FIG, 47c) denmstrated the sp: .eifcity of the assay, Thea high 24 specicity of the prCximitv-dependent enzyme channelin process is based on a configuration rcquing multpie detector-antibody bmding events on a common taret This COPIA format requiring coalizton of multiple doctor and capture an1tibodies is thereftec an ideal platform for multiplexed analysis ofi complex pathways. enoarait 25 f04961 COPA was utilized to profile HEi and HER2 in tumor tisses obtained by Ene needle aspiration (FNA) procedure from~ xenogra I -unuim Xeorafts wera derived fmm cell lines (MDA~MB-2, MDA-MB-43 and BT474) with vacrn degree of PbB-RTK expressionis (hmai, Y, e; aA, cancer Res. 42, 4394~4'398 Flmus st dMot Cel] BvA 7, 251^ (1987); Uherek, Qr at, Blood 100, 12650-73 (2002): Draga-wska, We al 30 Canr Res 2, 6069 (204) We detected low evltftpHER and piHERin MD haus 231 xenograft, high levei of pHER312 andA signilcamt level of pHEflRl (due to co-expression with amplified HER212) in FNA samupl es obtained from a BT1474 xeno graft Very low tHER 143 and ER2 activaton were detected EA obtained fItmf DAIB-435 enogra (a1d dw Ou fading from the en graftNA modey a orden±tb the parent cell ntfl 1NER2 profhe. dermstraiing tha ths method can be used tos detect activatior of EdtH receipts finsamples obtained fom minimnallyv invasive FNA proccdows. 5 neal perfermeee of COPIA Froyen tissue [014971 To t futher demnonstrate the clinical utility of COPIA, we: collected tissue 'aples fmm 26 stage 11 to 111 frozen BCA and 4 normal adjacent tisues va YNA procedure. Al prmary tmnor samples with high 1C score (3+) had high levels of HER2 expression and 10 showed a hWh degree of activation of HER Two out of twelve HER2-1HC positive samples (26135 and 20013) had lower tota-HER2 signal than other H ER2.-HC positive ampies, but bot had sabstantally higher sigd than HER2-nogative samples, All IlC-IER2-positive samples showed significant level o pHER2 signal The expression and activation of HER2 dtetd by our assay is concordant with the primary tum~or 11HC score (FIG. 47e anid FlG. 47f, PInteretingy, two of twelve primary IER2IHC positive paints also showecd a significant amount of pHERi with detectable total-HER! (12855) or within deteeiable total HERIU (128% This obsevadon suggests a Ith p of tyrosine kinSe inhibitors that can target both R TKs ay be more effective for a patient with th1s profile than therapies which target .ER RTK alone, All tissues with undetectable or low level prinry HRR2-HC 20 showed low levels of HER2 expression, but: some sample showed low but significant levels of HER2 activation. and this could have implications in patients resistant to hormonal themapy. Ca1elating Tumror Cells 10498} Recently CT s found in the blood. of metastatic cancer parents have been galling 2t significant attention as they provide an pportnity to perfonn non-invas5ve temporally~ rvant tumoor assessment (Cristofrmili. M4, ea cat ngl FJ Med 351, 781-91 (2004); Hayes, DtP et at. Clin. Cancer Rex 1:2> 4218-4224 (200);I Pacnn, K. et dat,. Cuts. co 28, 12081=215 (2008); Riethdor, S, et a! Cln Cancer Res, 1,92-8 (2007); Crson C reast Cancer 7.471~89 (2007)), in order to explore the 30 capability of COPIA to interrogate CT~s found in metastatic cancer patients, the technolo mst be demonstrated to be sensitive, specific, reproducible, standardized and related to clinical outcomtes, As RFU values do not provide toormttion per unit of cells, analytical 144 evauains for pathway expressioatiaiorfin were performed using standard cell lines with kown HER and HER2 expression. Algorthms converting RFU values into Computed Uhi (CUr a standard finiomal uit hsed on cell line controos were developed, For each zlide, a standard cemve consting ot serbily dilated ceU lysate was prepared fmm 5 lysates of cell ines MD~468 (HERI 0positve) and SKBr3 ( ER:-positve) as shown m FIG, 4Wa. Each wid as confidured to obtin quantitatie infornaion to accurately dtermite the level of HER and HER2 expression and the deree of phosphoryiation, as well as the level of cytkeratin (C). The limit of detection ('LD) value was determined to be less than 1CU ifr both piER (phosphoryiated HER1) and pER2 (phosphorylaed HER2) (FIG. 10 48b) Near I cell senshivity was observed for HE ,2 and CK, While the level of CK correates with the amount of CTCs in gen CTOS have different levels of CK (100 of MIDAMP468 cellbhowed less than 9.7 CU ad 10 SKBi3 cells showed 6.1 CU), In addition to the variation in the amount of CK expression, the type of CK expressed also varies in cb tumr with different eissu origin ja1haLA. N . a C&. I Oncoi 26, 2568 5 238i (2008)). Theref'ore -CK values rmay not serve as absoaute quantitative reference tor isolated CTCs, but Ti may be utilze as a tumor load idicatr for collected samnples from same pat nit aloo dhe course of the therapy. Slde? were scamned at multiple PIlT gain settings and slopes of the standard curve at each setting were determine. CI calclations were weighted proportonatly with values calculated. from standard curves with 20 a lowersope gveness weght> 0*499) A total of 87 whole blood sampltes, one each from 27 cancer patients and 60 healthy vhmteers 'were analyzed, The reference vahies for assays were determined based on data obtained &Rom 60 sampes from healthy control subjeels. Among 27 cancer samples analyzed 17 blood samples were bAmtaed w &m etastat bras a ner ptnts with 25 primary HER2,I3C negative status Samples with HER2 expression or actvation are shown in 110 48c, Up to 59% (10/17) ofiniial HER2 C negative samples showed evidence of 1RER12 activation in their CTCs, CTCs with HIR expu~ressionr were found in 7 out of 17 (41%) blood samples obtained loum me'-tastatic bresat cancer patients with primary HER2 negative status,. Here we were able to detect the HER2' activation even without appammn 30HER2 over-expression in 18h% (3/17) of CT~s from primary HER2-negative breast cancer patients. 6W% CTC samples (6/10) collected from relapsed BCA patients with primary HER2-ositive tumor still showed -HER2 expression. The level f pIHER' in rmary THC-> HER2+' (by IIC/FISH) patients who were st'll. on trastuzunab thenpy wre significant but 145 soewhat Lower than primary U4C-HER2-negadive patients whose 2Cr showed evidence of gaining H-Ei2-positive status, Patient A0C202 with extrenmely high HER2 expression (19A Ci Th wemad pHER2 1evel of 63U The pH HE R2 ia ai (63/194) which is slightly higher than typicsl pH ER2/HER2 ratio of unstimualated B1a4 type cls (21%) SKr3 cells have a higher degree ofbasai level phosphorylon of HER2 (FIS & WAIT LIISOU S I ON [O001 RTKs and downstream cefl-signaling proteins are major targets for therapeutic intervention in oncology, Interrogating the primary tumor in order to determiine potential 10 responsiveness to tageted therapy has become the standard of care, Full cha rerian of target expression, aotivation and domstreama cell signaling proteins is seldomx performed, however. Changes in the pattern of RT1K expesionU in tumor cell populations during tie time thame fromI initial diagnose to recurrence of metastatic disease is virtually never asses sed, A recent study by Meng el ai showed goodl concordanice between IhR2 gene Status in the primary tumor and in correspoMing CTCs only when sanplcs wer obtaied synchronously. However, CTCs fmm 24 relapsed patients with initial HER2 negative p rymor showed that 9 (37%) of 24 patients acquired HER2 amplification in their CTCs (Meng, S. et o, PNAS 10I 9393-98 0(04)), This study demonstrated discordance between primary and metastatic lesion of sufficicut signicace to alter disease mnana~gemret 20 Significant diiscrdance in HER2 over-expression between primary and tnetastatic sites has been reported using IHC in breast cancer (Zidan, I a? a, Br. 3 Cacer 93, 552456 (2005), and acquired I IR gene implication in CTCs vas confirmedl by anoter group (Hves D. et d , Ool 21 1111-117 (2002) This detseprofile shift may be due to teapeutic and other pressures on the heterogeneous tumor cell popudation of ma y cancers 25 that cause patterns of celignaling to evolve over time Our finding of tIER2-conversion may be due to lona . sedition of HER2-poshive cells within heterogeneous primary tumor cell population or gaining of genetic-capacty f'or over-expression (Le. gene ampli fiction> Regasdless of the mechanism behind H-ER~conversion, dt presence of HiiR22 in CTCs requires clinical attention, 30 jO5t0 COIA4 uties the tbrmation ofa unitine immno-camnplex equiring the co. lcalixation af two detector enzyme conjugamed-r1ibodies once target pteens are captued onthe microanrap-surfa'ce The channeling events between two detector enzymes with high hanover aurmberti s ( t/in ftGOX)aw ldd nn for HR?) in proimity enable the protndig of 146 the .R TK expression and activation in. a highly sensitive manner (Kltapper, Mvti. and Hacket DP YJwd Chem 238, 37363742 (1963A G o QwfL ef a, 3 Bio Cem. 9, 3 3934 (1%94W) T he analytical specitnty is greatly enhanced given the .requirement fo siruntaneous binding oF three different antibodies. This mnuiple xed COHJA cn in.iiate 5 ongitudinal monitoring of therapy progresion using rare CCs isolated from blood or other sources with limited numMe or turor zels such as FNA. This novel methd can be applied to qualify the express on and activation of other R TKs with transactivational pokendal and subsequent down stream cll-ignaling proteins from a single smple. 'Thbe ability to quantitate the trget protein activation state permits an additional evaluation of the signal 10 tramxductioni proteins beyond mere expression, potentially fourth r predicting the utility of various targeted therapicS. Furterm ore, assay component \pecifior additional targets can be added into muiplexed thrmat with a minimal disrption due to the requirement of multiple binding antibodies for specific signa! generation. W~.e havle demioustmted detection of' protein expression and actvation for at leasI GEI 4, C--MITX c-KIT, HER3, andp9$flER2 at 1$ a ingle digit Msllevel $05i2) Detectiont of any CTCs before initiation of firstline therapy ini patients with metastatic breast cancer predis for a poorer pro gresson t'ree survival and ovea 5allVstu corapared to patients without detectable CTC (Slaison, DJ. at at, Scince, 235, 17742 (1987); Cristofonili t a fJ S Agt .1 Med 351, 781 -91 (2004)), Although their 20 signrficance in mietastatic patients is still unknlownhL hving asenIsitIve method tonmonitor profil-shift in CT42s dw'rng the co-urse of therapy masy provide insight into the subsequent Course of the patient. This would greaty enhance the value of CTCbased therapy mnonitOrng over simple entaneration. As we can treat islae CT ~.s using reevn ligands, this technology can provide the "activation potential" tr CTXs in. their route to a poienttal 2$ me'tastatic site As the relationship of iT R2 gene status between the primary breast cancer and synchronous distant mietastasis has been re-portedi to be concordant by several groups and quite different by others (Zidan, 5 er aL,.B. I Ccurer 93 .52-556 (20y; 'Tanner Ma. et a , Cacer Re. 61, 5345-5348 (2001); Tapia C. e a, Or t Cancer Res 9, R1-3 (207) Gupka, NL. at at Arch. Patof Lab Mled 128, 974979 (2004)) it will be important to 30 ttenne the expression relationship between TK status in C'Cs versus rmary and m'astsdie kaions1 f0563j Regardless of §72 violation methods used,,enriched CTC saurpes typically contain at least 10 orhigher contanmnating blood ells. Performing gene expression analysis wuld 147 not be practical as non-TC related genesignaure is magnitudes tdgher. Because nur assay generates signat when binding partners are ini it provides a realsne dlinicai mteans to inve'stigaite care cells present in high non-target background een population Given that expssi and activation pamt of RTK and 5 subsequent downstream signd t sducinon pathway proteins change from the init workup to disease recunrence, cinicians may benent from a non-inasve Mai-te biopsy" and iongitudinai assessments that coulddprofile stuch changes, Therapeutic itervemtians coud be more rtonally 4atomtized to address these chsaging priles.Z' Ationlly, such assaysC can be used to fadlitate the development o new targeted therapies, I C MEODS Maltipleaed Microarray Printhng {5O4] 'The capture antibodies were prin ed on the surface of a mitroceilulose polymer coated glass slide (F AST®> Whta Florhamn Park, NJ), A contact mricroaray printer QArray, Genetix) was used to print capture ntiodies diluted ti X. FBS wth detergent. 15 The spot diameter was approximtely 17$ pa and printed slides were kept in a desiccated chamber at 4C, Each spot includes a taking dye and either a speed capture antibody (Ab) or controls printed in triplicates I serial dilitions. Approximately 500 pl f capture Abs were printed in implicate in serial dilution at concentrations of it , 9. 5mg/fm, and 02%mgtL (Th 48a) (plus 0,.12$ mg/mnl -- for alide in FHG 47a)i per each spot, Purified 20 mourse-g~s were taied as a negative control. Anytical calibration teacton were performed on 8 pads and imernal quality cantro! reactions on 2 pads, Each slide allows processing of up to 4 tunncew patient samples. tibody Conjgation and Pnrifeation [01051 Target specific antibodies nda corresponding detector enzymes were activated with 25 a bitctional cross l cik SUcnimidyl+(kaimiomethy) cyclohexane carboxylate (SMCC), and coupled to deximm to make anbodyt-decxtran-eazyme polymer conjugates, The cnaewas purified by HPLC uing a sizeexcusion coin. The antibody activities in the purified corugaes were dtectd by compel E ISA ad ezyme actvny was detected byv a functional assay specific for each detector enzyme, 1 48 Ce Line Sam ds 05061 r I DA4-3-68, T47iD and B'T474 cel hoes were obtained from ATCC, Cells were grownr in the ollowng growth media in 100 min tissue culture pates at 3C in 5% CO:: S (ur3 .McCoy s SA traedium wh 10% IBS, MUA-tM468: D4ME M, I 0% PBS, 5 B474: DMM. 108F FSa47h RPMI 1640, 10% PB3, 0,2 U/ml bovine insulin. Ce:ls were harveted at 70, 80% coiluenoy with gentl deiachmnwI process (trAin a ubsequent ntivatio) ad wn and washed with NU PS, Cei stimulation was performed with lOOPNM EGF or 20tM heregulin 0 or both in serm-&ree growth media for 5 md. Stimulated cells were washed ith X PBS and then were tysed 10 (JyS buffer: 50 mM Tris pIl 74, I30 mM NaC, 1% Triion X-100 and 200) mM Na3VO4 and kept on ice. for 30 tein Ciigea blood Sample5 IO5171 The clinical blood samples fhrn cancer patients as w ell as controlhealthy individu als were colbct according to the '1RB approve ed protocol tnformned consent was 15 gained A the use of all tie sampls, Each clinical pecimen was shipped to Promethes wthin 24 o0. samples were ocessed th sme dy and the resulting latest were stored at -804C All whole blood sapls were taken &onm adult subjects (>18 to<88 year) The samples were sourced fhrnm muliple CRO aites in Californaia. All1 cancer patients weare diagnosed according to current standard of care medical practice according to the RECIST 23 (Responsec Evuattion Criteria in SolTmrs) criteria, [05%)j The whole blood samples (N =27) weeotie r aint ihhsooia~ -confirmed breast carcinoma wit> regioreat lymph node or distant metIMsta e (Stage I or IV) regardless of their therapy sta at the time of blood sample collection. Subject i lbt Stage 111> breast cacer had rgional lymph nie staging of N 1, N!, or NY Metattic lesions were 2$ confimed with standard eshods (ag, whosebdy bonet scans, CT sOmn, PQ 1" scans, ecl Whole blond samples were cole Mcd by venpuncture from enrolled patients lit two tubes containing ElTA (purple top tubse) Colleced bod samples were shpped on thse day of collection at ambient lemperana. The identity of each patient was coded to preserve patient congtdentia~ify. 30 [059 Contol bood sampes frnm heath huteers 60) woet collected frm normal tidividaa between18and 7$ years assessed by a detailed medical istor to chde pro c o ccr th I er tous eo e di saseand a brcl physica.m inationiriluding blood prsaue Samdp ie Crlle mon nt j4151 The f a rozen breast cartissuesvwere purchased (ProteoGneo. CA and ILS Bio, $ MD), All paetst were caucasian with ducta carmoinma at stage I or IL RRR2 VHC status was provided for some samples, Tumor tissues were collected via FN A procedure by passing a 23 auge needle attached toan vad syringe S to 10 tius through t)C tissue equilibrated to room temperature, or by slicing of tlxzern tissues, FN Alissue samples weres lysed in 100 Ld Iysis buffer. AfTer incubating ont ice for 30 minutes, lysed samples were 10 eentifuged and the resuhlng supemaan af lysates was stored at -80oC, Xencgat models wAe constructed using human breast cancer cell ies (MDAMB-43$, MDA-MB-23 and B3T474) by subcutaneous injection into nude mice. When the trunor siote reached 400 nmn' un size, tissue samples were collected by passing a 23gauge needk attached to an evacuated syringe through each tumor S times CoLlected saes were lysed in 00 4 lys bufTe, 15 After incubaug on Re far 31 ama , ysed samples wre centrifuged and the resulting supernatant of states was stored at -804C CTC Sa mpies [0511t Peripheral blood was colce for CTCc evaluation. It5 ml of blood sames er draVn into 10-m evacuated EDTA tubs. Samples were maintaind at room temperatue 120 mailed ovaight, arnd pressed within 24 hours of colecTon. All CTC evaluadons were performed without knowledge of patent clinical status. The CellSearch System (ridex LLC1 Raritan, NJ) was used for immuno-uagneal CTC 0slatin according to the protocol previously described usn erfudts conjugated to, antibody againteihla elahso molecule (Feh, T. et a, Cilia Cner R~es. 8, 2073-84 (2002)), Enriched GT us from blood 2$ were simulated as described above COIWA [05921 ides were first rinsed two times with TBST (50 mM Tris 150 aM NaGVl 0.1% Twees-20, pH ,2-,4) before biking dhem with 80 ul ofWharman Blocking Bufer .hr at T or ON at 4C. After the b1ocking process, slides were washed 2 times with TPST 30 Serialy dihaed cell lysate controls in 80 m! of dilution buRr (2% SA/ 0,1 %1 triton X 100/TBS, pH '7,24 were added to nitrocellulose pads designated tr standards on the slide 150 and incubated for I hour at RT, Clinical samupes were also incubated in sWia hion in 80 Vi reaction volume. After the incubation slides were washed 4 times 3ain, each time, The detector antibodies (phosphorylated RT1K specific antibody-dextran-HRP, HER1a specific andb~ody-dxtrn-O, and 1HR2 specifieaniody-dextran-G~s in2% BiSA / 01% titoni X& 5 1 00 / TBS) were added in 80u of the reaction buffer and ncubated for 2 hours at RT. Unbound secondary detector antibodies were removed by washing with TBPST. The aetivation. state-independent antibodies were conjugated with channeling enzyme, GOC) and tire activation siate-dependent antibodies were kabeied with the signal amplification moiety; HIRP, When the GO is supplied with a substrate such as glucose, it generates hydrogen 10 peroxide ([B O). When the HRP is within the appropriate pm-rximuay to the GO, the Ft 0 is channeled to the fiRP where it frms a stable complex. The -HP 02 complex generates an amnpliiied signal using a tluorogenic substrate such as tyranmide to generate a rec&tiv'e tyramiide radical that covalendty binds nearbyvnuceophiic residues. In our assay, 80 pl of hiotin-tranmide (4001 pi/ml in ethanodl (Perkin Elmer Life cience) at S pg/mi in $0 nmi 15 glucosef PBS was added onto each pad and incubated for 15 main in the dark, Slides were then washed with TBST for 3mnin for 4 tioer, The activated tyramide is detected upon the addition of a signal-detecting reagent such as a strepraviin (SAP 4sbeled tluorophore. 80 gl of SA-Alexa647 (in PBS,. Invitrogen) at 0,5 pg/ni (1:4000 dilution) in 2% BSA/ 0.1% triton/TBSS for 40 main, pon a completion of' the incubation, slides were washed 4 times with 20 TBSLT Slides were then completely dried and kept in the dark uml scanning on mcrorra Western tBiting j0i 18 The cell lysates for each cell line were aliquoted into singhe use vials, The protein conceentration was determined by their DICA assay results, Samplos ware prepared with 2s: sample buffer containing p-mereaptoethanot. and after boiling for $ minutes and cooling to room temrperatrer the samples were loaded onto a NuPage 4-12% gel alongside a protein molecular weight ladder. Upon completion of electrophotesis, the separated proteins in. the gel were transferred to a nitrocelludose membrane (Invimoger, CA), The mnemrbrane was washed, blocked with 5% milk hiotto, and incubated with the primasy then secondary 30 antibodies hefore the detection process using NBT/BC1P, Data Analyste a15 014 Each slide was scanned at three phtoun iplier (PMT) gain settings to improve sensitivity and reduce thumpc-f saturation IPerkin Elmer ScanArray Express sotwatre was used for spot finding and signal qumtitation. The identifiW for each spOt were inpAted from a GenePIx Array List CAl) ile. The deldemitled sdy specific number for each 5 clinical sample on a slide was incorporated into the resulting data set, [0515) Background corrected signal intensities we're averaged for replicate spots prited in triplicate, The relative fluorescence value of the respective reagnent blanr was subtracted fsim each sample, Several quality criteria were used to fiter data from trther analysis including limits on the spot footprint coeficlent of variation for spot replicates, overall pad 10 backgro id and the intensity of the reagent blank, f0516J For each assay, a sgmoida standard curve was generated hom seven cnentratons ofsenally dilAted cell lysates prepared from cell lines MD46 (HER I positive) and SKr3 (HER2 positive Each curve was plotted as a fiction of signal inrensity vs. log concentration derived tnits CU (Compued Unit). The data were it to a five 1 parameter equation (P.L) by nonlinear regression (Ritz, C. and Streibig. . tJ. S-istrtkIl Sqfrvwre, 12, 1-22 (2005)) , sirwutaneously tting all three dilutions of the capture antibody Fitting was carried out using R, an open source statistical software packages (Dlevelopmtent Coxe leam. F: A !anguage and environment for statistical computing, R Foundation for Statistical Compuning, Vienna, Austria [SBN4 3~900051~074) UR1 jta nvK 20 grictg.R (200f)), To avoid ever paramneterization of the moathrematical model and thereby improve accuracy, four parameters were constrained, wile each didution was solved r an individual mfection point. This process was repeated fo each PMT gain setting of 45 50 and 60. This resulted in nine standuw curv generated per assay, from three dilutions of captaue antibody and three PM'T scans, The bailt-in redundancy in the assay allowed for one or more of the dilaution/scan combinations to be eliminated if the fit of the standard curve had an Rr less than 0.95 and thus improved subsequent predictions. An overview of the process for data reduction and data analysis is described in FIG, 49, and the standard curves generated are stown in FIG, 50a,. 5O517j 2 Caltdation (based on standard 3 utt The individadpredictions thorn ah of the standard curves (3 ape aibody ditions and3 PMgaineaning) were coined into a. inglefnal predicton, forch predcti, te slope of the point on the standard curve was calculated. This slope was taken wtogunits oni the ods , the uts in the denoiniznttor of the slo ar o Computed Uinits (CU). Second, a weighted average of the prdcin scluaeweethe weights were determined from the aiopes. Speetically, the weights wre summed, and each poin was given weightt cqual to its slope didd by dhe tota. slopes. This calcuat or one analyte is ilustMted as an example in F5 . 50b. Each assay was vacldated against prediction for knOWn controls. [05181 All pubisations and patet appicatins cited in this specific are herein incorporated by reference as Weach individual publication or pakm application were specifically and ndividualy IdjI ted to be incorporated byreferece, Abhough the foregoing invention bas been described in some detail by way of illustration and exple for 10 purposes of clarity of undstandng, it will be readily apparent to those of ordinary skill in the art in light of h teachmga of this invention that certain changes and m difcations may be made thereto without departing trm the spirit or scope of the appended claims, *15;

Claims (2)

1. 9. The method of clam 6, wherein the level of p95HR2 activation in the 2 cellular extract is at least S fhhgher than the reference activation level of p95HER2 10, The mef t iod of clain 6, wherein the presence of a similar or lower 2 level of HER2 or p95HER2 actvation in the ceihdlar extract compared to the reference 3 activation level of HER2 or p95HE1R2 indicates that the cell is sensitive to the compound. 1 11, The method of claim 1 wherein the eference activation level of HER2 2 or p95H}ER 2is obtained from a cell that is not treated with the compound. 1 12. The method of clain 1, wherein the method comprises determining the 2 activation level of both HER2 and p95HER2 in the celhdar extract 1 13, The method of clain 1, wherin the activation level of HER2 or 2 p95HLR2 comprises a phosphorylation level of HER2 or p951HEP2 1 14. The method of elaim 6, futrthr comprising deternining the activation 2 level of one or more additcal sinal transduction molecules in the elhiar extract I 15. The mefhod of claim 14, Wherein the one or more addit onal signal 2 transduction molecules is selected om the group consisting of EFR11 (HER1l), (lREE 3 11114, P13K, AKTMEK, PTEN SGK3, 4E1-BP 1ERK2 (MAPKI) ERK11 (MAPK3), 4 PDKl P70361 8K-3g Sh T M . c- METc-KIT, VEOFR .\ VEGFR2 VEGFR3,a 5 receptor dirner, and comnons thereof. 1 16. The method of claim 15, wherein the presence of a higher level of 2 HER3 activation in the ccltr extract compared to a reference activation vc I of' 1HER3 3 indicates that the cell is not sensitive to the compound. 1 17. The method of elaim 16 wherein the level of HER3 activation in the 2 cellhdar extract is at least 2 to fokd higher than fhe rerence activaton level of RER3I I M The method of claim.1, wherein the presence of a higher level of 2 P13K ictivat in ithe cellular eactm N scored to a refeence cdvation level of P13K 3 indicates that the cell is not sessitive to the compound 155 119 The method of claims 5, wherein the receptor dimer is selected fon 2 the grmp cormAsing of a p95iER2lHER3 heterodimer, HER2/HER2homodimer 3 HER2/EIR3 heterodnRer HERIER2 heterodimer HER2/HER3 heterdiner and 4 combinations thereof: 1 20 The method of d 9a , 19wherein the presence of a higher level of 2 p95dER2/HER3heterodimer activation in the cellular extract compared to a reference 3 activadon level of p95HFR21HER3 heterodimer indicates that the cell i. not sensitive to the 4 compound. 1 21 The mehod of laim I wherein the cell is a tumor cell. .1 21 The method of claim 21, wherein the tumor Cell is a circulating tumor 2 cell or a fine needle aspirate (FNA) cell obtained fiom a tamnor. 1 23, The method of claim 21, wherein the tumor cell is a breast cancer celt 1 24. The method of claim 1, wherein the cell is isolated rom a sample, 1 25. The method of claim 24. whereinrthe sample is obtained from a subject 2 with breas cancer, 1 26, The method of claim 24, wherein the sample is a whole blood, serum 2 plasa, or tumor tissue sample. 1 27. The method of clam 1, further comprising the step (e) of providing ite 2 result of the comparison obtaned in Ste> (d) to a usr in a readable format 28, The method of claim1 whrin detennining the activation level of 2 H.R2 or p95IER2. in step (c) comprises detctng a a Spho ylation level of HER2 or 3 p95HTER2 in the cellular extract with 'zn i specific for phosphorylated HER2 or 4 p95HER2.
2. 29. The meh od of claiM wherei determining the activation level of 2 HER2 in step (e) comprise: 156 3 (i) indubating the cellular extract with a dilition series of capture antibodies 4 specific for HB1T2 to fanta a phirality of captuad receptors wherein the capture antibodies 5 are restramied on a sold support 6 (ii) incubating the plurality of captured receptors wit detecton andbodies 7 comprsuing activation state-independent antibodies and activation state-dependent anrtibodies 8 specific for HER2 to form a phuraly of detectable captured receptors 9 w n the ativaton state-ndependentantibodies are labeled with a 10 faciiiating mot th actintion state~dependent antibodies are labeled with a fiat member 11 of a signal amplfication par and tie faciitating moiety generates an oidizing agent which 12 channels to and reacts wal the first member of the signalamplification pair; 13 ii) inubating the plurality o detectable captured receptors with a second 14 member of the signal amplfcation pair to generate an ampified signal; nd 15 (i) detecong the amupied sinal generated from the first and second 16 members of the signal amplification pair. 1 30, Ile method of claim 1,herein detering the activation level of 2 p9511ER2 in step (c) conprist 3 (i) incubang the cellar extract with a plrality of beads specific for an 4 extraelulnar dorai (R) binding region of fulw-ength HER?; 5 (II) removing the plualt o beds from the cellular extract thereby removing 6 fIull-iength HER2 to form a cellular tract devoid of fulllength hER2; 7 (i) incubating the ceular extent devaid of fullength HER2 wdih a di>ution 8 series of captre antibodies specific fo an intrmcelhudar domain (CD) hn region of full. 9 length HER2 to form a plurality of captured receptors, wherein the captue antibodies are 10 restrained on a solid support; II (iv) incubating the plurality of captured receptors with doeion antibodies 12 conpriing actvation state-independent antibodies and activation state-dependent antibodies 113 specific for an ICD binding region of filIen gth 1ER2 to form a plurality of detectable 14 zaptred receptors, i5 wherein the activation stare-independent antibodies are labeled with a 16 faciliating moiety, the ctivation statedependent antibodies are labeled with a first member 17 of a signa amplification pair, and the facilitating moiety generates an oxidizing agent wich 18 charmels to and reacts with the first member of the signal amplification park 157 19 (v) incubating the plurality of detectable captured receptors with a second 20 member of the signal amplifcationpair to generate an amplified Signal; and 21 (vi) detecting the am pne1 cd signal genetratedfom the first and second 22 members of the signal amplification pair 1 31 The method of dairm 30, wherein the plurality of beads specific for an 2 ECD binding region compares a sreptavidin-biotin pair werein the streptavidi is attached 3 to the bead and the biorin is attahed to an antibody. 1 32 The method of claim 31, wherein the antibody is specific for the ECD 2 binding region of2mlength HER2. 1 33, The method of daim 29 or 30, wherein the solid support is selected 2 fiom the group consisting of glass, plastic, chips, pIns, fibers, beads, paper, membrane, fiber 3 bundles. and combinations thereof 34 The method of claim 29 or 30, wherein. the capture antibodies are 2 restmned on thu solid uppor in an addressable array, 1 35 The method of claim 29 or 30, wherein the activation state 2 independent antibodies are directly labeed with the facilitating moiety. 1 36, 'The method of claim 29 or 30, herenthe aedvation srate-dependent 2 antibodies are drcirect labeled with he first member of the signal amplification pair, 1 37. The method of claim 29 or 30 whecin th Oativation state-dependent 2 antibodies are labeled with the first member of the signal amplfcion pair via binding 3 betw en a fitmember of a bidigpair cojgae o thd ctsroaae eun 4 antibodies and a second menber of the binding pair conjugated to the frst member of the 5 signal ampfitcation pair, 1 38, The method of claim 37, wherein the first member of the binding pair 2is biotin 1 39. The method of claim 37, wherein the second member of the binding 2 pair is wtrepta idin. 15S S40, The method of elam 29 or 30, wherein the facilitating moiety is 2 ghcose oxidase, 1 41, The method of claim 40r wherein the glucose oxidase and the 2 activation state-independent antibodies are cojugated to a sulfhydryl-activattied dextran 3 molecule. 1 42 The method of claim 41 wherein the sulflydryI-activated dextzran 2 molecule has a molecular weight of about4${IkDa. 43, The m ethod of claim 40, wherein the oxidizinga get is hydrogen 2 peroxide (i40i 1 44, The method of claini 43, wherein the first member of the signal 2 ampifcation pair is a peroxidase. 1 45, The method of claim 44, wherein the peroxidase is horseradish 2 peroxidase (HRP) 1 46, The method of claim 44 wherein the second member of the signal 2 amplification pair is a tyramide reagent, .1 47, The method of claim 46, wherein the tynride rezuent is biotin 2 tyramide. 1 48, The method of claim 47, wherein the amplified signal is generated by 2 peroxidase oxidization of the biotin-tyranide to produce. an activtxred tyramide. 1 49. The method of clain 48, wherein the activated tyranide is directly :2 detected 1 50. The method of claian 48 herein the activated tyramuide is detected 2 upon the addition of a sigrdetecting reagent 51 The method of claim 50,wherei the signal-detecting reagent is 2 streptavi din-bheled fluorophore5 1 59 1 51. The meut io SOclim , weenthe sigoa-deteczt ng titen :is 2 eaonibination. ofa swpatt~bidpuroxidase and. a chromnognic mean'at. 1 3. The methd of claim 52, wxheiia the &hmmoeio i reagent is 3 3',,54P-