pharmacodynamic response of the met/hgf...

Biology of Human Tumors

Pharmacodynamic Response of the MET/HGFReceptor to Small-Molecule Tyrosine KinaseInhibitors Examined with Validated, Fit-for-ClinicImmunoassaysApurva K. Srivastava1, Melinda G. Hollingshead2, Jennifer Weiner1, Tony Navas1,Yvonne A. Evrard1, Sonny A. Khin1, Jiuping Jay Ji3, Yiping Zhang3, Suzanne Borgel4,Thomas D. Pfister1, Robert J. Kinders1, Donald P. Bottaro5,W. Marston Linehan5,Joseph E. Tomaszewski6, James H. Doroshow6, and Ralph E. Parchment1

Abstract

Purpose: Rational development of targeted MET inhibitors forcancer treatment requires a quantitative understanding of targetpharmacodynamics, including molecular target engagement,mechanism of action, and duration of effect.

Experimental Design: Sandwich immunoassays and speci-men handling procedures were developed and validated forquantifying full-length MET and its key phosphospecies (pMET)in core tumor biopsies. MET was captured using an antibody tothe extracellular domain and then probed using antibodies toits C-terminus (full-length) and epitopes containing pY1234/1235, pY1235, and pY1356. Using pMET:MET ratios as assayendpoints, MET inhibitor pharmacodynamics were character-ized in MET-amplified and -compensated (VEGFR blockade)models.

Results: By limiting cold ischemia time to less than twominutes, the pharmacodynamic effects of the MET inhibitorsPHA665752 and PF02341066 (crizotinib) were quantifiable

using core needle biopsies of human gastric carcinoma xenografts(GTL-16 and SNU5). One dose decreased pY1234/1235 MET:MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60% to80%within 4 hours, but this effect was not fully sustained despitecontinued daily dosing. VEGFR blockade by pazopanib increasedpY1235-MET:MET and pY1356-MET:MET ratios, which wasreversed by tivantinib. Full-length MET was quantifiable in 5 of5 core needle samples obtained from a resected hereditary pap-illary renal carcinoma, but the levels of pMET specieswerenear theassay lower limit of quantitation.

Conclusions: These validated immunoassays for pharmacody-namic biomarkers of MET signaling are suitable for studyingMETresponses in amplified cancers as well as compensatory responsesto VEGFR blockade. Incorporating pharmacodynamic biomarkerstudies into clinical trials of MET inhibitors could providecritical proof of mechanism and proof of concept for the field.Clin Cancer Res; 22(14); 3683–94. �2016 AACR.

IntroductionThe receptor tyrosine kinase (RTK)MET [MET proto-oncogene,

RTK; hepatocyte growth factor receptor (HGFR)] is an important

drug target because of its roles in cancer progression, metastasis,and acquired resistance to EGFR inhibitors (1, 2). AberrantMET signaling can occur through ligand-dependent as well asligand-independent mechanisms (3). Consistent with multiplemechanisms ofMET dysregulation, there aremultiple therapeuticstrategies to target MET, including HGF or MET-neutralizingantibodies, decoy receptors, small-molecule tyrosine kinase inhi-bitors (TKI), and allosteric inhibitors of MET activation (4–7).Drugs from each mechanistic class are currently under clinicalinvestigation, both as single agents and in combinationwithothertreatments (8–11). Many of these clinical trials require patientselection based on immunohistochemical and/or gene copynumber assessment of MET (11, 12). However, these diagnosticassays cannot provide quantitative pharmacodynamic informa-tion (i.e., magnitude and duration of target modulation) to guideclinical development, nor can they distinguish between phos-phorylated epitopes of full-length MET and its degradation pro-ducts (13).

MET receptor activity is regulated by phosphorylation of anumber of sites including Y1234 and Y1235 in the activationloop, which are crucial for the regulation of kinase activity; thecarboxy-terminal Y1349 and Y1356 in the multifunctional dock-ing site required to recruit cytoplasmic signal transducers and

1Laboratory of Human Toxicology and Pharmacology, Applied/Devel-opmental Research Directorate, Leidos Biomedical Research, Inc.,Frederick National Laboratory for Cancer Research, Frederick, Mary-land. 2Biological Testing Branch, Developmental Therapeutics Pro-gram, Frederick National Laboratory for Cancer Research, Frederick,Maryland. 3National Clinical Target Validation Laboratory, Applied/Developmental Research Directorate, Leidos Biomedical Research,Inc., Frederick National Laboratory for Cancer Research, Frederick,Maryland. 4In Vivo Evaluation Group, Applied/DevelopmentalResearch Directorate, Leidos Biomedical Research, Inc., FrederickNational Laboratory for Cancer Research, Frederick, Maryland. 5Uro-logic Oncology Branch, NCI, Bethesda, Maryland. 6Division of CancerTreatment and Diagnosis, NCI, Bethesda, Maryland.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Ralph E. Parchment, Frederick National Laboratory forCancer Research, 1050 Boyles St., Frederick, MD 21702. Phone: 301-846-5427;Fax: 301-846-5206; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2323

�2016 American Association for Cancer Research.

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adaptors, and S975 and Y1003 in the juxta-membrane region,phosphorylation of which causes MET ubiquitylation and deg-radation (5, 8, 14, 15). On the basis of this understanding of METsignaling, we designed MET immunoassays to measure full-length, transmembrane MET protein and three key phosphospe-cies involved in its signal transduction: pY1234/1235-MET,pY1235-MET, and pY1356-MET. These assays were designed forthe measurement of wild-type MET, because mutations accountfor only 10% to 30% of all cancer subtypes where MET acts as adriver (compilation at http://www.vai.org/Met/Index.aspx; ref.16).After validating the immunoassays and specimen collection andprocessing methods in preclinical models, the possible clinicalsuitability of the immunoassays for studying MET in diseaseswithout gene amplification was evaluated in a case of MET-drivenhereditary papillary renal cell carcinoma (HPRC).

Materials and MethodsReagents

Purchased antibodies were qualified for use, including antigenaffinity–purified goat polyclonal anti-human MET extracellulardomain (catalog number AF276, R&D Systems); mouse mono-clonal anti-full-length MET (clone Met4, Van Andel ResearchInstitute, Grand Rapids, MI); mouse monoclonal anti-C-terminalMET (clone L41G3); and rabbit monoclonal anti-pY1234/pY1235-MET (clone D26, Cell Signaling Technology, Inc.). Tworabbit mAbs specific to pY1235-MET (clone 23111) and pY1356-MET (clone 7334) were developed under contract with EpitomicsInc. using phosphorylated peptide antigens corresponding toamino acid sequences surrounding these tyrosine residues (Sup-plementary Material). Recombinant MET (rMET) calibrator pro-tein was produced in HEK293 cells (Supplementary Material).Antibodies were biotinylated using Sulfo-NHS-LC-biotin(Thermo Fisher Scientific). Details of commercially available keyassay reagents are described in SOP341203 and SOP341206,available at http://dctd.cancer.gov/ResearchResources/ResearchRe-sources-biomarkers.htm.

Animal models and drug administrationAthymic nude mice (nu/nu NCr; Animal Production Program,

NCI-Frederick, Frederick, MD) were implanted with the humancancer cell lines U87 (glioblastoma); A549 (lung carcinoma);

MDA-MB-231 (breast carcinoma); HT-29 (colon carcinoma);or with GTL-16, MKN45, or SNU5 (all gastric carcinomas,MET-amplified) as described in ref.17. All cell lines were obtainedfrom theDivision of Cancer Treatment andDiagnosis Repository,NCI-Frederick and authenticated using AmpFLSTR Identifiler(Applied Biosystems).

MET inhibitors PHA665752 (NSC 748798-T), PF02341066(NSC 749769-Y, crizotinib), and tivantinib (NSC 758242);VEGFR inhibitor pazopanib (NSC 737754); and multikinaseinhibitor sorafenib (NSC 747971, lot #747971-U/3) were pro-vided by the Developmental Therapeutics Program, NCI(Bethesda, MD). Purity was established by proton–carbon NMR,HPLC, andmass spectrometry. Sorafenib was dissolved in DMSOfor in vitro studies. PF02341066 and pazopanib were adminis-tered by oral gavage in a saline vehicle and PHA665752 byintraperitoneal injections in a vehicle composed of 10% DMSOin saline. Tivantinib was administered orally in a PEG 400:20%vitamin E tocopheryl polyethylene glycol succinate solution(60:40) vehicle.

The NCI Animal Production Program, NCI-Frederick, is accre-dited by the Association for Assessment and Accreditation ofLaboratory Animal Care International and follows Public HealthService policy on the humane care and use of laboratory animals.All studies were conducted according to approved NIH AnimalCare and Use Committee protocols.

Xenograft biopsy and tumor quarter collection and extractpreparation

Specimen collection andhandling conditionswere adaptationsof those achievable in past NCI clinical trials (18, 19). Briefly, 18-gauge Temno Trucut needle biopsies were immediately flashfrozen in O-ring–sealed, conical-bottomed, screw-cap, 1.5-mLSarstedt cryovials. Tubes were sealed, returned to liquid nitrogen,and stored at �80�C until use. Whole xenograft tumors werecollected on the same schedule as tumor biopsies by standarddissectionmethods and cut into2 to4 equal pieceswithfine-pointscissors before flash freezing. All preclinical samples were frozenwithin 2 minutes of excision.

Tissue samples were processed by adding ice-cold Cell Extrac-tion Buffer (Invitrogen) and supplemented with PhosSTOP(Roche) and protease inhibitor tablets (Roche) to the frozentissue (0.35mLbuffer/biopsy and0.75mLbuffer/tumor quarter).Tissue was immediately homogenized with a PRO200 homoge-nizer with a Multi-Gen adaptor (PRO Scientific) and a 5-mmgenerator at the maximum setting for 10 seconds at 2�C to 8�C.The extract was vortexed and homogenization was repeated.Extracts were incubated at 2�C to 8�C for 60 minutes with orbitalshaking and clarified by centrifugation at 12,000� g for 5minutesat 2�C to 8�C. Cleared supernatant was aspirated and aliquoted.Total protein was measured by Bradford protein assay procedure(Bio-Rad).

Determination of mouse content of human tumor xenograftsMice were inoculated bilaterally with human tumor line cells

(1� 107) and tumor growthmonitored daily. One cohort ofmicewas grouped when tumors reached 100, 200, 400, 500, 800,1,000, 1,500, or 2,000 mg; the mean tumor size for eachweight-bin was determined and the tumors removed for analysis.A second cohort was euthanized 10, 14, 18, 22, 26, and 30 dayspostimplantation, irrespective of tumor size, and retrospectivelygrouped into 100, 200, 400, and 600 mg weight-bins. DNA from

Translational Relevance

Inhibiting aberrant signaling of the receptor tyrosine kinaseMET is a promising new therapeutic strategy for cancer treat-ment. Currently, several small-molecule inhibitors and ther-apeutic antibodies targeting MET are in various stages ofclinical development, but despite multiple clinical trials, clearevidenceof target engagement andmodulation in tumor tissueis lacking. We describe validated measurements of the phos-phorylation status of critical tyrosine residues in MET forassessing its pharmacodynamic response to targeted tyrosinekinase inhibitors. The ability to monitor full-length MET andits key phosphospecies in tumor biopsies and pMET specifi-cally in tumor xenografts will facilitate clinical studies thatcould link MET pharmacology to biology and clarify MET-based compensatory signaling in patients.

Srivastava et al.

Clin Cancer Res; 22(14) July 15, 2016 Clinical Cancer Research3684




one tumor quarter from each animal was analyzed formouse andhuman DNA content (20).

Xenograft ischemia studySNU5 tumor xenografts were staged to approximately 200 mg

(n ¼ 5/group). Needle biopsies were collected under anesthesiaand immediately flash frozen as controls. Tumors were excisedand quarters transferred to sterile normal saline maintained at25�C� 3�C (cold ischemia) or 37�C (warm ischemia) for 1, 2, 3,4, 5, 8, 10, 15, 30, or 60minutes in temperature-controlled salinebefore being flash frozen. All flash-frozen samples were stored at�80�C, and lysates were processed within 2 weeks of collection.

Fit-for-purpose MET inhibitor studiesFit-for-purpose studies result in validated pharmacodynamic

assays and provide drug mechanism of action information. Todemonstrate assay fit-for-purpose in preclinical models, micebearing GTL-16 and SNU5 xenografts were randomized whentumors reached 200� 25mm3 in size and dosed daily for 8 to 10days with vehicle, oral PF02341066 (6.25, 12.5, or 25 mg/kg), orintraperitoneal PHA665752 (25or 50mg/kg). In a separate study,mice with SNU5 xenografts were staged to 200 mm3 and treatedfor 8 days with vehicle (daily), pazopanib (100 mg/kg daily),tivantinib (200 mg/kg daily), or combinations of pazopanib(daily) with 2 dose levels of tivantinib (200 mg/kg daily or200 mg/kg twice daily). Extracts of core needle biopsies collected4 hours after tivantinib administration on day 8were analyzed forMET, pMET, and hypoxia-inducible factor 1, a subunit (HIF1a;ref. 21).

Core needle biopsies of HPRC tumorMultiple 18-gauge needle biopsies were collected from a sur-

gically resected tumor from a patient with HPRC (UrologicOncology Branch, NCI, Bethesda, MD) within 2 minutes ofresection and immediately flash frozen. The patient gave writteninformed consent for study inclusion and was enrolled in an NCIInstitutional Review Board–approved protocol. Study design andconduct complied with all applicable regulations, guidelines, andlocal policies.

MET immunoassay procedureThe full-length MET immunoassay measures the total levels of

full-length MET in tumor tissue samples, irrespective of phos-phorylation status. An affinity-purified goat polyclonal antibodyagainst human MET extracellular domain (catalog numberAF276) was used to coat 8-well Nunc MaxiSorp strips overnightat 2�C to 8�C with 1 mg/mL buffer (0.1 mol/L sodium carbonate/bicarbonate buffer pH 9.6, 100 mL/well). Wells were blocked for 2hours with 1� PBS, 0.5%mannitol, 0.2% glycine, and 0.2% BSA,and strips were assembled on 96-well plate frames, freeze dried,and sealed in desiccated pouches (6-month stability). For theimmunoassay, 100 mL each of purified rMET calibrator, controls,and tumor lysates (at protein concentrations of 10–50 mg/mL)prepared in assay buffer [1� PBS/casein (BioFx) containing Phos-STOP, protease inhibitors, and 0.1% Triton X-100 (Roche)] wereadded to the AF276-coated wells. The rMET assay calibrator wasprepared for the dynamic range of the assay. Plateswere incubatedat ambient temperature (25�C � 3�C) on an orbital shaker (600rpm) for 1 hour to capture MET. After three washes with assaybuffer, 100 mL/well 200 ng/mL biotin-conjugated mouse mono-clonal MET antibody (clone L41G3) was added to the plates and

incubated for 1 hour at 25�C � 3�C, followed by a second washand the addition of 100 mL/well 200 ng/mL streptavidin poly-HRP conjugate (Pierce). After 30-minute incubation with theHRP-conjugate, plates were washed 4 times and 100 mL Super-Signal ELISA Pico Chemiluminescent Substrate (Pierce) wasadded to each well. Plates were read within 10 minutes using anInfinite 200Microplate ELISA Reader (Tecan).MET immunoassaycalibration curves were analyzed on GraphPad Prism, andunknown values were calculated using 4-parameter curve fitting.

Three different pMET immunoassays were developed forthe pharmacodynamic studies: pY1234/1235-, pY1235-, andpY1356-MET (Supplementary Material). The pMET immunoas-say procedures were similar to the full-lengthMET immunoassay,except that rabbit mAbs specific to pY1234/1235-MET (cloneD26, Cell Signaling Technologiy), pY1235-MET (clone 23111),or pY1356-MET (clone 7334) were conjugated to biotin and usedas reporter antibodies for the immunoassay. The pMET assaycalibrators were prepared for the dynamic range of each assay.Assay control samples were prepared by combining GTL-16,SNU5, A549, MKN45, or U87 xenograft tumor lysates to achievelow, medium, and high MET concentrations at 10 to 50 mg/mLtotal protein concentrations.

MET immunofluorescence analysisHT-29, GTL-16, and A549 cancer cell lines were treated with

DMSO vehicle (0.1% w/v), 100 nmol/L PF02341066, or 100nmol/L sorafenib for 24 hours at 37�C. Cells were fixed in 10%neutral-buffered formalin (Sigma-Aldrich) for 24 hours, pelleted,and then embedded in paraffin. After 5-mm sectioning, antigenretrieval with Bond Epitope Retrieval Solution 2 (at 100�C for 10minutes) and immunofluorescent stainingwere performedon theBOND-MAX Autostainer (Leica Biosystems). For antigen detec-tion, 10 mg/mL primary antibody (anti-pY1235–MET, clone23111) was followed by 10 mg/mL goat anti-rabbit AF488 (LifeTechnologies). Immunofluorescence microscopy was performedon core needle biopsies of a resected humanHPRC tumor stainedwith 5 mg/mL anti-full-length MET (clone Met4), followed by 10mg/mL anti-mouse AF660 (Life Technologies). Image acquisitionand analysis were performed on a wide-field fluorescent, confocalmicroscope (Nikon 90i, Andor Camera, NIS Elements Software).

Statistical analysesRegression analysis and descriptive statistics, including means,

SDs, coefficients of variation (CV), one-wayANOVAanalyses, andStudent t testswere conductedwithMicrosoft Excel andGraphPadPrism (v3.04). The 95% confidence interval was significant at a¼0.05 for a two-sided t test.

ResultsDesign and development of sandwich immunoassays formeasuring key phosphoMET species

Tyrosines 1234, 1235, and 1356 mediate MET signal transduc-tion via phosphorylation; so many MET-targeted agents aredesigned to reduce MET phosphorylation at one or more of thesesites. To study the pharmacodynamics of such drugs, a sandwichimmunoassay was designed to capture MET via its extracellulardomain and then probe the captured MET for a particular phos-pho-epitope (pY1234/1235, pY1235, or pY1356) using specificmAbs (Fig. 1A). The capture antibody essentially did not cross-react with recombinant extracellular domains of the two most

Measurable Pharmacodynamic Biomarkers of MET Activation

www.aacrjournals.org Clin Cancer Res; 22(14) July 15, 2016 3685




likely cross-reactive human tyrosine kinases, RON (macrophage-stimulating 1 receptor; MST1R) and EGFR. At 10-fold excessconcentration (4–400 pmol/L) over rMET (0.4–40 pmol/L),cross-reactivity with RON was <5%, and EGFR cross-reactivitywas not detectable (data not shown). The pY1235-MET specieswas measured using a new rabbit mAb (clone 23111) thatrecognizes the pY1235 epitope independent of Y1234 phos-phorylation status, and its high specificity was demonstrated byWestern blot analysis using synthetic peptides, rMET, cell lysates,and mouse tissues (Supplementary Fig. S1A and S1D). Unlikethe specificity of D26 antibody for the dual-phosphorylatedpY1234/pY1235 epitope, the mAb produced by clone 23111showed no cross-reactivity with the cytoplasmic domain of RONor with this MET epitope when phosphorylated only at Y1234 orwhen tyrosine 1235 was replaced by aspartic acid (Supplemen-

tary Fig. S1A and S1B). It stained the plasma membrane of MET-expressing cell lines in an HGF-dependent manner that wassensitive to MET inhibitors, but not sorafenib (Fig. 1B). A newrabbit mAb recognizing the pY1356 epitope of MET (clone7334) was also generated for use in the immunoassay (Supple-mentary Fig. S1C and S1D).

Analytic validation of the sandwich immunoassaysUsing full-length, purified rMET as calibrator, the dynamic

ranges of the calibration curves for each assay were established:0.3 to 40 pmol/L for full-length and pY1234/1235-MET assaysand3.125 to200pmol/L for the pY1235- andpY1356-MET assays(Fig. 1C). Assay sensitivities were 1.5, 1.5, 7.8, and 15.6 fmol/mgprotein, respectively, and the assays did not significantly crossre-act with mouse MET species (Supplementary Materials).

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Figure 1.Development of immunoassays for full-length MET and its key phosphorylated species. A, schematic of the MET immunoassays: a capture antibody (catalognumber AF276) specific to the extracellular domain of MET binds and traps MET from tissue lysates, then four different reporter mAbs (clone D26 to pY1234/1235,clone 23111 to pY1235, clone 7334 to pY1356, and clone L41G3 to the C-terminus) are used to detect phosphorylated and full-length MET. B, specificity ofanti-pY1235-MET (clone 23111; developed in this study) is shown by immunofluorescence staining of formalin-fixed, paraffin-embedded GTL-16, HT29, andA549 cancer cells treated in vitro with 100 nmol/L PF02341066 or 100 nmol/L sorafenib for 4 hours. C, representative calibration curves from the full-lengthMET (0.3–40 pmol/L), pY1234/1235-MET (0.3–40 pmol/L), pY1235-MET (1.5–200 pmol/L), and pY1356-MET immunoassays (3.25–200 pmol/L). The samerMET protein is used as a calibrator in all four immunoassays, and levels have been converted to log pmol/L. D, MET levels in four different human cancer celllines routinely used in MET preclinical xenograft studies; two separate GTL-16 and U87 xenograft samples were used. Cell lysates were diluted to 31.25 to2,000 ng/well in 100 mL volume; MET levels were back-calculated to fmol/mg total loaded protein. RLU, relative luminescence units.

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TheMET immunoassayswere subjected to a rigorous validationprotocol for analytic performance using clinically relevant tumorsampling procedures (core needle biopsy of tumors in mice) andspecimen preparation procedures (Supplementary Material).Dilution linearity studies showed that tumor extracts could bediluted up to 8-fold without affecting assay performance (Sup-plementary Table S1). Spike-recovery experiments of rMET inxenograft extracts established immunoassay accuracy. Recoveryranged from 78% to 116% for full-length MET, pY1234/1235-MET, and pY1235-MET assays and 86% � 29% for pY1356-MET(mean � SD; Supplementary Table S1). Intraplate (n ¼ 20) andinterday (n ¼ 5) variation were evaluated using three xenografttumor extracts assayed at minimum of 5 different days by twooperators; intra-assay CV was <10% and interassay CV was <14%(Supplementary Table S1). After reducing the procedures andoperating parameters of the validated assay to standard operatingprocedures (SOP), assay robustness was formally demonstratedby SOP-driven assay transfer from the development to the clinicaltesting laboratory as described previously (18, 22).

The validated assay was capable of quantifying full-lengthMET in extracts of GTL-16, A549, MDA-MB-231, and U87xenograft tumors over a wide range of protein loads per well(Fig. 1D). Full-length MET was detected over a range of 0.05 to1.79 fmol/mg protein, and as expected, the MET-amplifiedmodel (GTL-16) contained the highest levels (1.31–1.79fmol/mg protein). A least significant change (LSC) calculation,which combines both technical variation of the assay andbiologic variation of the biomarker (determined by analyzingmultiple quadrants of xenografted SNU5 tumors), established a45% change as the minimal effect level required to demonstratea drug effect (Supplementary Material).

Biopsy handling to control preanalytic variablesPreanalytic variables (specimen handling, shipping, and stor-

age procedures) can have a significant impact on the reliability ofbiomarkermeasurements in the laboratory, andphosphoproteinsinvolved in dynamic responses of signaling pathways are noto-riously labile during specimen collection due to ischemia andother factors (23–25). The stability of full-length MET and itsphosphospecies was characterized in biopsies of SNU5 xenograftssubjected to increasing ischemia time, which was defined as thetotal time needed for core needle biopsy sampling, specimenhandling, and flash freezing. MET levels in core needle biopsysamples frozen immediately after collection from anesthetizedanimals (defined as the zero time point) were set as baseline(100%). Both pY1235- and pY1356-MET levels decreased by>60% during 15 minutes of cold ischemia (25�C � 3�C) andcontinued decreasing over the next 15minutes (P < 0.05; Fig. 2A).During 15 minutes of warm ischemia (37�C), both pY1235- andpY1356-MET levels decreased by >80% (P < 0.05; Fig. 2B). ThepY1234/p1235-MET species exhibited similar degradation underthese conditions (data not shown). In contrast, full-length METlevels were relatively stable for up to 60 minutes of cold ischemiabut decreased significantly at 37�C (Fig. 2A and B).

A follow-up study evaluated the stabilities of pMET and full-length MET during 8 minutes of cold ischemia. After just 3minutes of cold ischemia, pY1234/1235-MET levels decreasedby >50% (P < 0.05; Fig. 2C). On the basis of Western blot analysisresults, loss of full-length pY1235-MET signal during cold ische-mia was predominantly due to loss of the pY1235-MET epitope,plus a small amount of degradation of full-length MET (the

appearance of N- and C-terminal fragments; Fig. 2D). Stabilitiesof the pY1234/1235-MET and pY1356-MET analytes were similar(data not shown). Therefore, core needle biopsies frozen within 2minutes of collection will yield valid pMET assay results.

Pharmacodynamics ofMETRTK inhibitors in gastric carcinomaxenograft models

Mice with GTL-16 tumor xenografts, a MET-amplified model,were treated daily with the MET inhibitors, PHA665752 orPF02341066, and sampled at clinically relevant time points (4or 24 hours) after drug administration. The 25 and 50mg/kg/daydose levels of PHA665752 slowed the growth ofGTL-16 xenografttumors and achieved tumor stasis after 8 to 10 days of treatment(Fig. 3A, study days 17–19). Four hours after the first dose, therewas a dose-dependent reduction in the pY1235-MET:MET ratio of62% and 80%, respectively (Fig. 3B), effect sizes that exceeded theLSC threshold of 45%. Over the next 20 hours, the pY1235-MET:MET ratio partially to fully recovered, depending upon dosage.Additional daily doses of 50 mg/kg PHA665752 significantlydecreased the pY1235-MET:MET ratio, whereas the 25 mg/kgdose produced inconsistent changes (Fig. 3B). The ratio ofpY1356-MET:MET also declined after the first dose, although itrecovered to normal levels within 20 hours (Supplementary Fig.S3A). Therefore, the tumor stasis observed with this agent anddosage regimen was associated with intervals of modest reduc-tions in the pY1235 and pY1356 targets, followed by targetrecovery. The absolute level of full-length MET also decreased by40% to 50% within 10 days of treatment with either vehicle ordrug (Supplementary Fig. S3B).

As reported previously (10), treatment of GTL-16 xenograftswith daily oral PF02341066 at the 25 mg/kg dose level achievedstasis of GTL-16 xenografts by the seventh day of treatment (Fig.3C; study day 11). Only this dose level reduced the pY1235-MET:MET ratio (by 65%) 4 hours after treatment, and this ratiorecovered completely during the next 20 hours (Fig. 3D). Addi-tional daily doses of PF02341066 further reduced the pY1235-MET:MET ratio, so molecular target control 4 hours after eachdose was achievable throughout the daily treatment period. TheSNU5 model exhibited a similar dose-dependent pharmacody-namic response to PF02341066 and again pointed to greaterpharmacodynamic responsiveness of pY1235 than pY1356 fol-lowing TKI therapy (Supplementary Fig. S4).

A second study of GTL-16 xenografts treated daily withPF02341066, but with denser tumor sampling to more fullycharacterize the pharmacodynamic response, confirmed that onlythe 25mg/kg/day dose level achieved amolecular target responseafter the first dose. However, there was a cumulative, dose-depen-dent pharmacodynamic effect on the pY1235-MET:MET ratiosuch that by day 4, both the 25 and 12.5 mg/kg dose levelsproduced pharmacodynamic responses 4 hours and 12 hoursafter drug administration, although only the higher dose main-tained this pharmacodynamic response for the entire 24-hourdosing interval (Fig. 4A). The pY1356-MET:MET ratio decreasedafter 4 and 12 hours but fully recovered by 24 hours at both doselevels (Fig. 4B). Again, full-lengthMET levels decreased by 40% to50%by treatment day 10 in both drug- and vehicle-treated groups(Fig. 4C).

This preclinical modeling suggests that greater control of METsignaling than the intermittent 80% reduction in the pMET:METratio observed here will be needed to achieve regressions of MET-amplified tumormodels. These data also indicate the importance






of using the more consistent pMET:MET ratio instead of absolutelevels of particular pMET species as pharmacodynamic endpoints,at least in preclinical models where mouse cell infiltration ofhuman tumor xenografts over time diluted the absolute level ofhuman MET per microgram–extracted protein (SupplementaryFig. S5).

Fit-for-purpose modeling of the first-in-human application ofthe assay

The first clinical application of the validated assay is assessmentof tumor pharmacodynamic response during a phase I clinicaltrial of the VEGFR/platelet-derived growth factor receptor(PDGFR) inhibitor pazopanib alone and in combination withthe MET inhibitor tivantinib (ClinicalTrials.gov NCT01468922).The trial is testing two hypotheses: (i) MET will be upregulated aspart of a compensatory response to VEGFR/PDGFR inhibition bypazopanib and (ii) tivantinib will blunt that upregulation byinducing degradation of total MET (26). To model this trial, mice

bearing SNU5 xenografts were treated with vehicle or the drugsindividually or in combination, and the tumors were sampled onday 8 using 18-gauge biopsy needles to replicate the samplingplanned for the clinical trial. Neither single-agent pazopanib nortivantinib affected full-length MET levels (Fig. 5A). However,treatment with pazopanib, but not tivantinib, increased bothpY1235-MET:MET and pY1356-MET:MET ratios compared withvehicle (Fig. 5B and C). Twice daily tivantinib (200 mg/kg)combined with pazopanib returned the pMET:MET ratios tovehicle-treated levels, whereas daily tivantinib treatment (200mg/kg) caused an intermediate effect (Fig. 5B and C). To deter-mine whether the underlying changes in MET levels were asso-ciatedwith hypoxia induced byVEGFR/PDGFR inhibition,HIF1alevels weremeasured using a validatedHIF1a immunoassay (21).Pazopanib-induced increases in pMET:MET ratios were accom-panied by a 46% increase in HIF1a levels (P < 0.05), whereas theaddition of tivantinib returned HIF1a to vehicle-treated levels(Fig. 5D).

Figure 2.Stability of full-length MET and pMET in core needle biopsies of xenografted SNU5 tumors. Core needle biopsies were incubated in saline solution for 0(baseline, 100%), 1, 15, 30, or 60minutes of cold ischemia (A) orwarm ischemia (B). C, follow-up study of stability of full-length and pY1234/1235-MET in core biopsiesduring 0, 1, 2, 3, 4, 5, and 8 minutes of cold ischemia. For all graphs, error bars are mean � SD, n ¼ 4–6. Asterisk (�) denotes P < 0.05 from baseline byStudent t test. D, Western blot analysis of extracts of core biopsies after 0, 1, 15, 30, or 60 minutes of cold ischemia probed with the indicated antibodies (Ab).

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MET and pMET levels in clinical cancer without METamplification: HPRC

The baseline levels of MET and pMET in human tumor modelswithout MET amplification (Fig. 1D) are low, lying near the assaylower limit of quantitation; therefore, drug-induced decreases inpMET:MET ratios cannot be quantified in these models. Toaddress the question whether MET and pMET levels in preclinicalmodels and in clinical cancer without substantial MET amplifi-cation are similar, we evaluated core needle biopsies of fiveregions of a resected HPRC tumor harboring a germline METmutation (H1112R). HPRC is characterized by germline trisomyof chromosome 7, frequent expression of two copies of mutatedMET and one copy of wild-type MET (27, 28), and decreased

activation threshold of the kinase activity and enhanced trans-forming activity (15); so, HPRC was a nonamplified disease inwhich pMET quantitation seemed likely. As expected, full-lengthMET levels ranged from 0.073 to 0.368 fmol/mg protein (Supple-mentary Table S2), similar to nonamplified models. However,pY1234/1235-MET was measurable in only two of five cores(0.037 and 0.041 fmol/mg), and neither pY1235-MET norpY1356-MET were detectable in any core specimen.

Immunofluorescence staining of sections of the HPRC coreswith the capture antibody from the immunoassay (AF276, anti-MET extracellular domain) confirmed the presence of MET in theHPRC cells and localized it primarily to the plasma membrane(Fig. 6A). Western blotting revealed that the undetectable level of

Figure 3.Reduction of GTL-16 tumor growth and MET phosphorylation by MET RTK inhibitors. A, tumor volume of GTL-16 tumor xenografts in mice treated withPHA665752 at daily doses of 0, 25, and 50 mg/kg i.p. starting 10 days after implantation; n ¼ 5–28 per dose per time point. B, the intratumoral pY1235-MET:METratio during 10 days of daily treatment with PHA665752; n ¼ 5–6 per dose per time point. Tumor samples were analyzed at 4 and 24 hours after dose 1 and4 hours after doses 3 (D3), 8 (D8), and 10 (D10). C, tumor volume of GTL-16 xenografts in mice treated with PF02341066 at daily doses of 0, 6.25, 12.5, and25mg/kg orally starting at 4 days after implantation. Error bars aremean� SD; groups n¼4–30per dose per time point. D, the pY1235-MET:MET ratio during 10 daysof daily treatment with PF02341066. Tumor samples were analyzed at 4 and 24 hours after dose 1 and 4 hours after doses 3 (D3), 6 (D6), and 10 (D10). Mean� SD,groups n ¼ 2–6 per dose per time point. For A and C, �, P < 0.05 and �� , P < 0.001 compared with vehicle group by unpaired Student t test. For B andD, the dotted line indicates the LSC in pMET:MET ratio from the vehicle-treated group of 45%; changes larger than this are attributed to drug effect.






pY1235-MET in the immunoassay was due in large part to theassociation of the epitope with a truncated C-terminal 50 kDaMET fragment (Fig. 6B), which is too small to contain the N-terminal domain required for capturing full-length MET in the

validated sandwich immunoassay. A small amount of full-lengthMET containing the pY1235 epitope was detectable by Westernblotting, but detection required loading the gel lane with 25 to50 mg total protein, which exceeds the amount of protein that can

Figure 4.Reduction of GTL-16 tumor growth and MET phosphorylation by PF02341066. A, pY1235-MET:MET ratio and pY1356-MET:MET ratio (B) during dailytreatmentwith PF02341066. Tumor sampleswere analyzed at 4 and 24 hours after dose 1, then 4, 12, and 24 hours after dose 4 (D4), 12 hours after dose 5 (D5), and 4,12, and 24 hours after dose 10 (D10). Mean � SD, all groups n ¼ 2–6 per dose per time point. The dotted line indicates the LSC in pMET:MET ratio from thevehicle-treated group of 45%; changes larger than this are attributed to drug effect. C, absolute levels of full-length MET plotted over time following daily doses of12.5 and 25 mg/kg PF02341066 or vehicle; data normalized to extracted protein.

Figure 5.Treatment of SNU5 gastric cancerxenografts with the VEGFR inhibitorpazopanib activates MET. Full-lengthMET, pMET:MET ratios, and HIF1alevels were measured on day 8 oftreatment with vehicle, tivantinib orpazopanib alone, or the combinationof pazopanib þ tivantinib [daily (QD)or twice daily (BID)] in combination.Absolute levels of full-length MET (A),pY1235-MET:MET ratio (B), andpY1356-MET:MET ratio (C). Thedottedline indicates the LSC in pMET:METratio from the vehicle-treated groupof 45%; changes larger than this areattributed to drug effect. D, HIF1alevels were measured in extractsgenerated from xenograft tumorquadrants. All graphs plotted mean �SD, all groups n ¼ 5–6 per dose pertime point. Single asterisk (�) P < 0.05compared with vehicle group.

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be used in the immunoassay while maintaining assay linearity(Fig. 1D). Interestingly, the antibody against pY1234/1235-MET(cloneD26) recognizedmainly full-lengthMET (Fig. 6B),which iswhy it was detectable in the immunoassay.

DiscussionBiochemical signaling from activated MET drives both prolif-

eration and migration of malignant cells (1, 2), and MET can beactivated not only by conventional ligand binding of paracrineand autocrine HGF, but also by several ligand-independentmechanisms. Transphosphorylation of Y1235 in the kinasedomain initiates signaling, and subsequent phosphorylation ofY1349 and Y1356 in the SH2-bindingmotif transduces signaling,with phosphorylation of Y1356 critical for growth factor recep-tor–bound protein 2 docking that may link MET to the RASpathway (29, 30), analogous to EGFR signaling. Several pharma-cologic classes ofMET inhibitors target these key phosphorylationevents, so the extent of phosphorylation of the critical tyrosineresidues should be a highly informative pharmacodynamic bio-marker of molecular drug action. Importantly, we demonstratedthat the phosphorylation status of the key tyrosine residues in full-length ligand-responsiveMET is quantifiable using a dual-epitopesandwich immunoassay platform, with newly generated mAbsspecific for pY1356 and for pY1235 (independent of phosphor-ylation status of the adjacent Y1234 residue), that will be madeavailable to the research community via the DevelopmentalStudies Hybridoma Bank (http://dshb.biology.uiowa.edu/).Also, to obtain valid assay results, cold ischemia time must belimited to 2minutes to preserve the phosphorylated analytes. For

preclinical pharmacodynamic studies in xenograft models, ahuman pMET:MET ratio normalizes pMET to total MET andmitigates the confounding effect of mouse cell infiltration astumors grow (20).

Daily dosage regimens of twoMET TKIs achieved tumor controland pharmacodynamic responses in GTL-16 and SNU5 humantumor xenograft models of poorly differentiated gastric carcino-ma driven by MET amplification (31, 32). Tumor control of GTL-16 with PHA665752, a highly selective, ATP-competitive inhib-itor of MET RTK activity (33), was associated with a 60% to 80%reduction in the pY1235-MET:MET ratio 4 hours after the firstdose, but the ratio fully recovered by 24 hours and was lessresponsive to subsequent days of therapy. Treating GTL-16 withPF02341066, a potent, orally bioavailable, ATP-competitiveinhibitor of MET catalytic activity (34), achieved tumor controlat theonly dose level that elicited apharmacodynamic response (aconsistent 65%–90% reduction in the pY1235-MET:MET ratiothroughout the 24-hour dosing interval). The pY1356-MET:METratio was consistently less responsive to drug therapy than thepY1235-MET:MET ratio in both tumor models and with bothMET TKIs, suggesting GRB2 signaling is more difficult to inhibitthan MET kinase activity.

Importantly, 60% to 90% reductions in the pY1235-MET:METratio were associated only with tumor stasis, but not regression.Therefore, a greater than commonly seenMET pharmacodynamicresponse, that is, the magnitude and/or duration of pY1235-METinhibition, or the additionof pY1356-MET inhibition (also linkedto tumor regression; ref.35) must be needed to induce tumorregression in MET-amplified models. Clinical benefit from MET-directed therapy has been limited to patients with high baseline

Figure 6.MET assessment in core biopsies of asurgically resected HPRC tumor. A,immunofluorescence staining of asection from a formalin-fixed, paraffin-embedded core needle specimen of aresected HPRC tumor. Merged andindividual fluorescent images of N-terminus MET staining with AF276antibody (pink; primarily membrane)and nuclear staining with DAPI (blue).Magnification, 40�. B, tissue extractsof HPRC core biopsies sequentiallyanalyzed by Western blot analysisusing antibodies against pY1235-MET(clone 23111), pY1234/1235-MET (cloneD26), N-terminal MET (AF276), andC-terminal MET (L41G3). Anti-pY1235-MET primarily recognizes a truncated,C-terminal form of MET, whereas anti-pY1234/1235-MET recognizes mostlyfull-length MET. In addition, a minorprotein band >170 kDa was detectedwith the MET N-terminal specificantibody, which is not recognized bythe other MET antibodies. The METN-terminal–specific antibody alsorecognizes a truncatedN-terminalMETfragment. Sample loadwas 25 to 50mgprotein per gel lane. The numbers "2"and "3" indicate thespecimennumbersdescribed in Supplementary Table 2.






MET expression (36–39), but these assessments were made with-out a validated pharmacodynamic biomarker for activatedMETorknowledge of the effect of warm ischemia on full-length METdegradation. Therefore, the trials to date unfortunately representmissed opportunities to understand clinical pharmacodynamicresponses elicited by currentMET inhibitors that could guide theirfuture clinical development (40). The newly developed andvalidated assays described here are suitable for defining thepharmacodynamic response associated with tumor regressionboth in preclinical models and human patients with MET-ampli-fied cancers. The analytic performance of the assays is sufficient forconducting small, fast phase 0 clinical trials of various dosageregimens of investigational MET inhibitors to discover how tomaintain over 90% target suppression during dosing intervals(41, 42). From our error modeling, considering both biologicvariability and assay precision, clinical pharmacodynamic studydesigns based on longitudinal patient sampling should requiredecreases in pMET:MET ratios of at least 45% (the LSC value) toattribute them to drug effect.

In addition to MET amplification with its relatively high base-line pMET:MET ratios, assay fitness-for-purpose was also dem-onstrated in a MET-induction setting during compensatoryresponses to tumor hypoxia. Induction of MET signaling convertsa tumor from low to measurably high MET and pMET levels,which become targets of MET TKI therapy. We found that phar-macologic targeting of tumor vasculature in SNU5xenograftswithpazopanib, a multikinase inhibitor, induced the hypoxia bio-markerHif1a and increasedpY1235-MET:MET andpY1356-MET:MET ratios. Although twice daily treatment with tivantinib, anallosteric inhibitor of MET (6, 7, 26) with some effects onmicrotubules (43–45), did not reduce baseline MET or pMETlevels, it nevertheless blocked pazopanib-induced HIF1a METsignaling. Several molecular mechanisms could explain whytivantinib blocked pMET increases induced by pazopanib, includ-ing blocking the microtubule dependent coclustering of METand VEGFR2 or prolonging PTP1B (protein tyrosine phosphatase,non-receptor type I) inhibition ofMET (43, 44, 46). Clinical trialsof MET/VEGF inhibitor combinations [pazopanib/tivantinib(NCT01468922) and bevacizumab/tivantinib (NCT01749384)]provide an opportunity for correlative pharmacodynamic studiesto determine whether this compensatory mechanism operates inclinical cancers, and the preclinical modeling of one of these trialsdemonstrated that the validated pMET immunoassay is a suitabletool for these studies.

This newly developed and validated immunoassay for phar-macodynamic biomarkers of MET molecular response is a robusttool for understanding and optimizing pharmacologic control ofboth amplified and induced MET signaling, and it is ready fortraining-based transfer to the research community by the NCI(http://dctd.cancer.gov/ResearchResources/ResearchResources-biomarkers.htm). Phosphorylation of Y1235 is the proposed firststep of MET signaling upon ligand binding, and the specificity ofthe new mAb for phosphorylated Y1235 independent of thephosphorylation status of the adjacent Y1234 residue will beuseful for teasing apart the role of these neighboring tyrosinesduring initiationofMET signaling andmolecular response toMETTKIs. Importantly, the anti-pY1235 mAb we generated (clone23111) exhibited much greater specificity than the anti-pY1234/1235mAb (clone D26), which even recognized a recom-binant GST-MET protein harboring a Y1235D substitution. Our

studies found that pharmacodynamic responses of the pY1235and pY1234/1235 biomarkers did not always correspond. Fur-thermore, both pY1235 and pY1234/1235 epitopes were identi-fied in full-length MET extracted from an HPRC tissue sample,whereas the pY1235 epitope was mostly present in a 50 kDaMETfragment. Similarly, complex phosphorylation profiles have beenfound in non–small cell lung cancer, in which only 25%ofMETþcases were also positive for pY1234/1235-MET (47). The pY1235-specific antibody couldbe a key reagent in anewdiagnostic test forselecting the right patients to receive MET-targeted therapiesbecause it substitutes the phosphorylated MET kinase domain(48) in place of elevated MET, mRNA, gene copy number, colo-calized proteins, or multiply phosphorylated epitopes (49, 50).However, unlike the validated immunoassay, cross-reactivity toMET fragments with undefined biologic significance (27) willconfound the reported immunohistochemical tests of MET sig-naling (26, 37, 51–53).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerThe content of this publication does not necessarily reflect the views or

policies of the Department of Health andHuman Services, nor doesmention oftrade names, commercial products, or organizations imply endorsement by theU.S. Government.

Authors' ContributionsConception and design: A.K. Srivastava, M.G. Hollingshead, J.J. Ji, R.J. Kinders,D.P. Bottaro, J.E. Tomaszewski, J.H. Doroshow, R.E. ParchmentDevelopment of methodology: A.K. Srivastava, T. Navas, S.A. Khin, J.J. JiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.K. Srivastava, M.G. Hollingshead, J. Weiner,T. Navas, S.A. Khin, J.J. Ji, T.D. Pfister, D.P. Bottaro, W.M. Linehan,J.H. DoroshowAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.K. Srivastava, J. Weiner, T. Navas, Y.A. Evrard,J.J. Ji, Y. Zhang, T.D. Pfister, D.P. Bottaro, J.E. Tomaszewski, R.E. ParchmentWriting, review, and/or revision of the manuscript: A.K. Srivastava,M.G. Hollingshead, T. Navas, Y.A. Evrard, J.J. Ji, Y. Zhang, T.D. Pfister,R.J. Kinders, D.P. Bottaro, W.M. Linehan, J.H. Doroshow, R.E. ParchmentAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.K. Srivastava, Y.A. Evrard, S.A. Khin, J.J. Ji,Y. Zhang, S. Borgel, T.D. Pfister,W.M. Linehan, J.E. Tomaszewski, J.H.DoroshowStudy supervision: A.K. Srivastava, M.G. Hollingshead, Y. Zhang, J.E. Tomas-zewski, R.E. ParchmentOther (conceptualized the project with others in DCTD, assigned to theDCTDPharmacodynamic Group at NCI-Frederick, andmanaged on aweeklyto monthly basis during project team meetings): J.E. Tomaszewski

AcknowledgmentsThe authors thank Drs. Andrea Regier Voth (Leidos Biomedical Research,

Inc.) and Joseph Shaw (Kelly Services) for knowledgeable and skilled medicalwriting support in the preparation of this manuscript.

Grant SupportThis project has been funded in whole or in part with federal funds from the

NCI, NIH, under contract no. HHSN261200800001E.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 25, 2015; revised February 23, 2016; accepted February23, 2016; published OnlineFirst March 21, 2016.

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2016;22:3683-3694. Published OnlineFirst March 21, 2016.Clin Cancer Res Apurva K. Srivastava, Melinda G. Hollingshead, Jennifer Weiner, et al. Validated, Fit-for-Clinic ImmunoassaysSmall-Molecule Tyrosine Kinase Inhibitors Examined with Pharmacodynamic Response of the MET/HGF Receptor to

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