Direct Evidence of Target Inhibition with Anti-VEGF, EGFR ...clincancerres.aacrjournals.org/content/clincanres/early/2015/06/17/1078-0432.CCR-14... · pathway (using VEGFR2 as the

Personalized Medicine and Imaging

Direct Evidence of Target Inhibition withAnti-VEGF, EGFR, and mTOR Therapies in aClinical Model of Wound HealingJingquan Jia1, Andrew E. Dellinger2, Eric S.Weiss3, Anuradha Bulusu2, Christel Rushing2,Haiyan Li4, Leigh A. Howard1, Neal Kaplan1, Herbert Pang2,5, Herbert I. Hurwitz1, andAndrew B. Nixon1

Abstract

Purpose: In early clinical testing, most novel targeted antican-cer therapies have limited toxicities and limited efficacy, whichcomplicates dose and schedule selection for these agents. Con-firmation of target inhibition is critical for rational drug devel-opment; however, repeated tumor biopsies are often impracticaland peripheral blood mononuclear cells and normal skin areoften inadequate surrogates for tumor tissue. Based upon thesimilarities of tumor and wound stroma, we have developed aclinical dermal granulation tissue model to evaluate novel tar-geted therapies.

Experimental Design: A 4-mm skin punch biopsy was used tostimulate wound healing and a repeat 5-mm punch biopsy wasused to harvest the resulting granulation tissue. This assay wasperformed at pretreatment and on-treatment evaluating fourtargeted therapies, bevacizumab, everolimus, erlotinib, and pani-tumumab, in the context of three different clinical trials. Total and

phosphorylated levels VEGFR2, S6RP, and EGFR were evaluatedusing ELISA-based methodologies.

Results: Significant and consistent inhibition of the VEGFpathway (using VEGFR2 as the readout) was observed ingranulation tissue biopsies from patients treated with bevaci-zumab and everolimus. In addition, significant and consistentinhibition of the mTOR pathway (using S6RP as the readout)was observed in patients treated with everolimus. Finally,significant inhibition of the EGFR pathway (using EGFR asthe readout) was observed in patients treated with panitumu-mab, but this was not observed in patients treated witherlotinib.

Conclusion: Molecular analyses of dermal granulation tissuecan be used as a convenient and quantitative pharmacodynamicbiomarker platform for multiple classes of targeted therapies. ClinCancer Res; 1–11. �2015 AACR.

IntroductionFor the clinical development of novel targeted therapies, it is

critical to validate that targets of interest are being inhibited.Manytargeted therapies may not have dose-limiting toxicities, or evencommon non–dose-liming toxicities, which could otherwiseguide dose and schedule selection. Furthermore, preclinical mod-els often donot accurately reflect the human setting for a variety ofreasons. A key consequence of this limitation is the difficulty ofusing these models to identify a specific pharmacokinetic param-eter that can be used to guide dose selection in patients. Aside

from dose selection, pharmacodynamic markers can also beuseful to evaluate the downstream consequences of target inhi-bition, which may affect drug sensitivity, resistance, and toxicity.

In patients with cancer, paired pretreatment and on-treatmenttumor biopsies remain the gold standard for the evaluation oftarget inhibition and the downstream consequences of that inhi-bition. However, repeat biopsies carry significant risks and costs.In addition, only a minority of patients will have tumors amend-able to serial biopsies. The small size of such biopsies, variablestromal contributions, and heterogeneity within and amongtumor lesions, can all complicate interpretation of tumor-basedpharmacodynamic studies. For these reasons, surrogate tissueshave been extensively evaluated for biomarker assessments,including plasma and serum, circulating peripheral mononuclearcells (with orwithout ex vivo stimulation), quiescent skin, andhairfollicles.However, the relevance of these tissues is often uncertain.The assessments of skin and hair follicles are also often furtherlimited by the amounts of tissue provided and the use of largelysemiquantitative IHC methods.

To address these limitations, we have developed a dermalwound model for the assessment to targeted therapies in patients(1). This model initially uses a punch biopsy to stimulate woundhealing. After one week, the wound is filled with granulationtissue, which can then be harvestedwith a repeated punch biopsy.This granulation tissue provides a sufficient amount of materialfor molecular analyses by ELISA or PCR, although each method-ology needs significant optimization for these types of analyses. In

1Department of Medicine, Division of Medical Oncology, Duke Univer-sity Medical Center, Durham, North Carolina. 2Department of Biosta-tistics and Bioinformatics, Duke University Medical Center, Durham,North Carolina. 3Temple University School of Medicine, Philadelphia,Pennsylvania. 4Department of Medicine, Center for MusculoskeletalResearch, University of Rochester Medical Center, Rochester, NewYork. 5School of Public Health, Li Ka Shing Faculty of Medicine, TheUniversity of Hong Kong, Pok Fu Lam, Hong Kong SAR, China.

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

Corresponding Author: Andrew Nixon, Division of Medical Oncology, Depart-ment of Medicine, Duke University Medical Center, 395 MSRB1, 203 ResearchDrive, Box 2631, Durham, NC 27710. Phone: 919-613-7883; Fax: 919-668-3925;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-2819

�2015 American Association for Cancer Research.

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addition, the surrounding dermis displays a highly reproduciblepattern of vascularization that can be quantified, consistent withthe role of angiogenesis in wound healing.

This model is safe, convenient, low-cost, and can be usedrepeatedly in the context ofmost clinical trials. Granulation tissue,as opposed to quiescent skin, provides a potentially clinicallyrelevant surrogate tissue because tumor stroma and wound stro-ma exhibit many similarities, a topic which has been extensivelyreviewed (2). Indeed, tumors have been compared with "woundsthat do not heal" (3). Granulation tissue is highly proliferativeand angiogenic, which makes this approach particularly wellsuited for drugs with antiangiogenic properties. Wound healingand skin toxicities have been reported formany targeted therapies,including VEGF, mTOR, and EGFR inhibitors (4, 5).

Previously, we have shown that this model could detect theantiangiogenic effects of multiple antiangiogenic agents (6). Wehave further optimized the methods for imaging the dermisaround the wound and developed new methods to allow char-acterization of key signaling proteins to be performed on theharvested granulation tissue. This report describes the feasibilityof using this wound model to investigate the pharmacodynamicmodulation of key antiangiogenic protein targets in response to aseveral distinct therapies. Although we have shown that ourmodel can be used in a semiquantitative manner to evaluatewound angiogenesis, we now show that the dermal granulationtissue can be used for quantitative assessment of key signalingpathways under physiologic conditions and during treatmentwith targeted agents. Here, we evaluate this model in a series ofthree related phase I and II clinical trials that evaluated doubletand triplet combinations of either bevacizumab, everolimus,erlotinib, or panitumumab, agents that target the VEGF, mTOR,andEGFR, signaling pathways, respectively. Biomarker substudiesin these trials were specifically designed to help assess the phar-macodynamic impact of each of these drugs on their respectivetargets.

Patients and MethodsClinical trials

The parent phase I and phase II clinical trials that includedthe current biomarker substudies have been previously reported(7–9); characteristics of those patients who participated in the

biomarker studies and details of the treatment regimens are listedin Supplementary Tables S1–S3, as well as in Fig. 1. All patientsparticipating in these biomarker studies were treated at DukeUniversity Medical Center (Durham, NC) and signed informedconsent for the therapeutic study, which included the describedbiomarker analyses. All studies were approved by the DukeUniversity Medical Center Institutional Review Board and theDuke Comprehensive Cancer Center Protocol Review Commit-tee, andwere registered on clinicaltrials.gov. The eligibility criteriafor the parent treatment studies have previously been reported.The doses for each agent used in this biomarker study are listed inSupplementary Tables S2 and S3. Bevacizumab was dosed at itsstandard higher dose (10mg/kg i.v. every 2 weeks) in all patients.Everolimus was dosed at its standard dose of 10 mg orally dailywhen combinedwith bevacizumab. Because of increased levels ofrash and mucositis seen with anti-mTOR and anti-EGFR combi-nations, when these agents were combined in these studies, thedoses of everolimus (5 mg daily or 5 mg three times per week),erlotinib (75 mg orally daily), and panitumumab (4.8 mg/kg i.v.every 2 weeks) were below their respective monotherapy doses,although these doses remained within the standard dose reduc-tions used for these agents. Sequential skin biopsies were requiredfor each of these treatment studies unless the patient had acontraindication to a skin biopsy (such as active skin infectionor skin condition) or where drug toxicity, disease progression, orinter-current illness lead to a treatment interruption that wouldhave compromised the biomarker analysis.

Skin biopsy procedures and vascular scoring. Skin biopsies wereobtained using previously reported methods (1). Briefly, beforetreatment, a 4-mm skin punch biopsy (Fray Products Corp.) wascreated to stimulate granulation tissue formation followed by a5-mmbiopsy of the healing wound (granulation tissue) collected7 days later. Briefly, the patient's skin was locally anesthetizedwith 1% lidocaine with epinephrine at a ratio of 1:100,000mixedwith sodium bicarbonate (8.4%) followed by either a 4-mmstimulation or 5-mm granulation punch biopsy. Topical antibi-otic ointment followed by a nonocclusive bandage was applied toeach wound. Tissue biopsies were embedded in optimal cuttingtemperature (OCT) compound and then immediately snap fro-zen in liquid nitrogen and stored at �80�C. Patients were givenwritten andverbalwound care instructions. The granulation tissuebiopsies (5 mm) were harvested at baseline (before any treat-ment) and after at least one week of treatment with the respectivetargeted agent(s). The time points for each biopsy are listed in Fig.1. On treatment time-points were expected to reflect near steadystate levels of each drug while minimizing patient travel andtreatment delays. Dermal neovascularization at the woundperiphery was evaluated using a digital camera with a specialdermatologic adapter (Heine Dermatophote). All of the imageswere scored using an ordinal 0–4 scoring system of woundvascularization, with each wound field divided into 12 clockhours. Each image was scored by two independent observers,blinded to treatment status and image timing, and average vas-cular scores (AVS) were obtained for each time point (1).

Protein extraction from granulation tissue biopsies. Tissue sampleswere thawed at room temperature and removed from the OCTonce softened. The samples were washed in ice-cold PBS andimmediately transferred to Fast Prep Homogenization tubespreloaded with ice-cold lysis buffer, a modified RIPA buffer

Translational Relevance

Direct and quantitative evidence of target inhibition byantiangiogenic agents is needed to monitor the pharmacody-namic effects of targeted therapies in patients. In this article, wehave examined the modulation of downstream moleculartargets of VEGF, mTOR, and EGFR inhibitors in granulationtissue obtained at baseline and on-treatment from patientsenrolled across three different clinical trials. Our results dem-onstrated significant and consistent inhibition of the targetedpathways after treatment in eachof the clinical trials examined.Granulation tissue biopsies are less expensive and more read-ily accessible than tumor biopsies. These features make gran-ulation tissue biopsies a practical and reliable tissue source forfurther exploration of the pharmacodynamic effects of noveltargeted therapies.

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containing 20 mmol/L HEPES, 154 mmol/L NaCl, 2 mmol/LMgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100,proteinase inhibitor cocktail (Roche, cat# 04693150001), andphosphatase inhibitor cocktails I and II (Sigma, cat# P2850 andP5726). Tissues were homogenized using Bio101 ThermoSavant FastPrep FP120 (Qbiogene, Inc) tissue homogenizer at4�C. Lysates were subsequently centrifuged at 16,000 � g at 4�Cto remove unbroken cells, nuclei, and other particulates.

Protein analysisProtein lysates were analyzed using the Meso Scale Discovery

platform (MSD). Human KDR kit (Cat# K151BOC) and Phospho(Tyr 1054) VEGFR2 Whole Cell Lysate kit (Cat# K151DJD),Phospho(Ser240/Ser244)/Total S6RPWhole Cell Lysate kit (Cat#K15139), and Phospho (Tyr1173)/Total EGFRWhole Cell Lysatekit (Cat# K15104D), were used to evaluate VEGFR2, mTOR, and

EGFR pathways. All assays were performed in duplicate using 50to 100 mg of total protein according to manufacturer's recom-mendations and read on MSD sector imager 2400 instrument(MSD, Cat# R92TC-2).

Statistical analysisFrequency tables were generated for the demographic vari-

ables and median and range were provided for the relevantvariables. To evaluate on-treatment changes, L-ratio was calcu-lated using the formula Log2 (posttreatment level/baselinelevel) for each marker. To determine the significance of L-ratiochanges, Wilcoxon signed rank tests were used to analyzechanges in total VEGFR2, phospho-VEGFR2, total EGFR, phos-pho(Tyr1173) EGFR, total S6RP, phospho(Ser240/Ser244)S6RP expression, as well as AVS. Waterfall plots for on-treat-ment changes were illustrated. Boxplots for percentage change

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from baselines were illustrated. Results with significant P valueswere further corrected for multiple testing with Benjamini–Hochberg FDR. The correlation between (i) the change inanalyte levels of the various treatment regimens and (ii) AVSand inhibition of VEGFR2 was evaluated using Spearman's rankcorrelation.

ResultsPatient characteristics and study cohort description

The number of the patients with molecular biomarker analysisand their demographics across each study are summarized inSupplementary Table S1. No significant wound-healing compli-cations from wound biopsies occurred on any of these trials.

In the phase I BEE study, a total of 38 patients with a variety ofsolid tumors were evaluable for biomarker analysis. Thesepatients were treated on four biomarker cohorts: (i) everolimusmonotherapy for 2weeks before the addition of bevacizumab anderlotinib ("Ev-BEE" cohort, n ¼ 9); (ii) erlotinib monotherapyfor 2 weeks before the addition of bevacizumab and everolimus("Erl-BEE" cohort, n ¼ 9); (iii) bevacizumab plus everolimusdoublet therapy ("BE" cohort, n¼ 15); and (iv) bevacizumab pluseverolimus plus erlotinib triplet therapy ("BEE" cohort, n ¼ 5).Full-thickness dermal granulation tissue biopsies were taken atbaseline, end of themonotherapy lead-in period (day 14), and 35days post-doublet or triplet therapy. All patients who receivedbevacizumab, everolimus, and erlotinib treatments concurrentlywere analyzed as one group, referred as "BevþEvþErl," whetherthey received this triplet therapy initially (n ¼ 5), or whether thetriplet was received after everolimus monotherapy (n ¼ 8) anderlotinib monotherapy (n ¼ 7) lead-ins.

In the phase I BEP study, a total of 18 patients with a variety ofsolid tumors were evaluable for biomarker analysis. Thesepatients were treated in four biomarker cohorts: everolimusmonotherapy for 2 weeks before the addition of bevacizumaband panitumumab ("Ev-BEP" cohort, n ¼ 1), bevacizumab pluseverolimus doublet therapy for 2 weeks before the addition ofpanitumumab ("BE-BEP" cohort, n ¼ 5), everolimus plus pani-tumumab doublet therapy for 2 weeks before the addition ofbevacizumab ("EP-BEP" cohort, n ¼ 4), bevacizumab plus ever-olimus plus panitumumab triplet therapy ("BEP" cohort, n ¼ 8).For those cohortswho received amonotherapy or doublet therapylead-in, dermal granulation tissue biopsies were taken at baseline,the end of lead-in therapy (day 14), and 42 days after BEP triplettherapy. For the "BEP" cohort, dermal granulation tissue biopsieswere taken at baseline and 28 days posttreatment. Because of thesmall number of patients available for biomarker analysis in eachcohort of the BEP study, the lead-in time point was not analyzed.All patients who received bevacizumab, everolimus, and panitu-mumab treatments concurrently were analyzed as one group. Thisincludes 8 patients in the "BEP" cohort, 3 patients in the "BE-BEP"cohort, 2 patients in the "EP-BEP" cohort, and1patient in the " Ev-BEP" cohort.

In the phase II Bev-Ev study, 40 patients with refractory met-astatic colorectal cancerwere evaluable for biomarker analyses. Allpatients received bevacizumab plus everolimus doublet therapy;there was no monotherapy component or cohort in this study.Dermal granulation tissue biopsies were taken at baseline and 28days posttreatment. The time points for drug administration andbiopsy collection for these three clinical trials are summarizedin Fig. 1.

Inhibition of total and phospho VEGFR2 proteinTotal VEGFR2 levels were significantly reduced by bevacizu-

mab treatment given in combination with everolimus (Fig. 2Cand Fig. 2F), with everolimus plus erlotinib (Fig. 2D), and witheverolimus plus panitumumab (Fig. 2E; P < 0.05). Phosphor-ylation of VEGFR2 was also significantly inhibited by bevaci-zumab treatment given in combination with everolimus(Fig. 3F), and in combination with everolimus plus erlotitinb(Fig. 3D). However, phospho VEGFR2 was not significantlyinhibited by BevþEvþPmab treatment (Fig. 3E). Because of theconcern of the small sample size of each treatment group andtherefore limited statistical power, a combined analysis wasdone using data pooled from all three trials where every patientwho had received bevacizumab during the course of the studywas analyzed together as one group, referred to as "All Bev Pts."For those patients who have received bevacizumab from thelead-in phase, only the final on-treatment biopsy was includedfor the statistical analysis. Both total and phospho VEGFR2were significantly inhibited in the "All Bev Pts" group (Figs. 2Gand 3G; P < 0.001), and the average percent inhibition wasapproximately 50% for total VEGFR2 and 25% for phosphoVEGFR2 (Figs. 2H and 3H). Even though the VEGFR2 assayswere normalized to total protein, the specific cellular compo-sition of the biopsy samples were not measured due to thelimited size of the biopsies; thus these experiments cannotdetermine whether the reductions in total VEGFR2 might bedue a reduction in the number of endothelial cells, a reductionin VEGFR2 production per endothelial cell, or both mechan-isms. Monotherapy treatment with everolimus, which is knownto have potent antiangiogenic activity, also led to a significantloss in total VEGFR2 expression (Fig. 2A; P ¼ 0.002). However,phospho VEGFR2 levels were not significantly downregulatedby everolimus monotherapy (Fig. 3A). Erlotinib monotherapydid not appear to affect VEGFR2 expression or phosphorylation(Figs. 2B and 3B)

Taken together, these data demonstrate direct target inhibitionof VEGFR2 by the combination of bevacizumab and everolimustreatment. This combination decreased receptor expression anddecreased the phosphorylation state of the receptor. Althougheverolimus monotherapy reduced total VEGFR2 expression, thecombination of bevacizumab plus everolimus did not appear toincrease the level of inhibition over that seen with everolimustreatment alone (Fig. 2H).

Inhibition of total and phospho S6RP protein expressionp70S6 Kinase (S6K1) and 4EBP1 are two well-characterized

downstream effectors of mTOR activity. Activated S6K1 phos-phorylates S6 ribosomal protein (S6RP) to promote mRNAtranslation (10). The level of phosphorylated (Ser240/244) S6RP(pS6RP) has been shown to reflect the activation of S6K1 (11).Preclinical studies suggested that S6RP is a reliable measure ofmTOR blockade (12, 13). For these reasons, total and phospho(Ser240/244) S6RP protein levels were used as markers of mTORinhibition.

Everolimus monotherapy did not significantly change totalS6RP protein expression (Fig. 4A). Erlotinib alone did not havea significant effect on either total (Fig. 4B) or phospho levels ofS6RP (Fig. 5B). However, total S6RP protein expression wassignificantly decreased by everolimus treatment in combinationwith bevacizumab (Fig. 4F), with bevacizumab plus erlotinib(Fig. 4D) or with bevacizumab plus panitumumab (Fig. 4E;

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P < 0.05). As a direct target of mTOR pathway activity, phosphoS6RP protein levels were significantly reduced by everolimustreatment given alone (Fig. 5A), in combination with bevaci-zumab (Fig. 5C and 5F), in combination with bevacizumabplus erlotinib (Fig. 5D), or in combination with bevacizumabplus panitumumab (Fig. 5E; P < 0.05). All patients whoreceived everolimus treatment on one of the three clinical trialswere also evaluated as one group, referred to as "All Ev Pts."Both total and phospho S6RP protein expressions were signif-icantly reduced (Figs. 4G and 5G), and the average percentinhibition observed was about 50% for total S6RP and 85% forphospho S6RP (Figs. 4H and 5H). Taken together, our datademonstrate direct target inhibition of the mTOR pathway by

everolimus treatment, either alone or in combination withbevacizumab and EGFR inhibitors.

Inhibition of total and phospho EGFR protein expressionTotal EGFR (tEGFR) protein levels and phosphorylated

(Tyr 1173) EGFR (pEGFR) were evaluated to monitor the activityof erlotinib and panitumumab in the granulation tissue model.Interestingly, erlotinib monotherapy did not appear to signifi-cantly reduce either total EGFR or phospho EGFR levels, althoughour study was not powered to detect modest levels of inhibition(Fig. 6B and 6F). Similarly, no significant decrease in total orphospho EGFR protein levels was observed with erlotinib treat-ment in combination with bevacizumab and everolimus (Fig. 6C

Figure 2.Changes in total VEGFR2 levels frombaseline. A–G, each waterfall plotrepresents a specific treatmentregimen in each trial. The y-axisrepresents the log fold change in totalVEGFR2 protein level from baseline.Each line along x-axis represents anindividual patient. H, percent changein total VEGFR2 protein levels frombaseline in response to the differenttreatment regimens. Ev, everolimusmonotherapy; Erl, erlotinibmonotherapy; BE, bevacizumab þeverolimus double therapy; BEE,bevacizumabþ everolimusþ erlotinibtriple therapy; BEP, bevacizumab þeverolimus þ panitumumab tripletherapy; BEE_BEP, BEE and BEPregimen combined; Pooled, alltreatment regimen combined.






and 6G). However, panitumumab treatment, in combinationwith bevacizumab and everolimus, significantly inhibited bothtotal and phospho EGFR levels (Fig. 6D and 6H; P < 0.001). Thisresult is consistent with the fact that panitumumab competitivelyinhibits EGFR ligand binding, promotes receptor internalization,and leads to tyrosine kinase phosphorylation (14).

In addition, we examined the correlation across the change inanalyte levels after the various treatment regimens. We comparedall phospho proteins among themselves, and all total proteinsamong themselves. Spearman's rank correlations were calculatedand P values were Bonferroni multiple testing corrected. Allmarkers were positively correlated with one another, as all corre-

lations had a positive rho value (correlation coefficient). AfterBonferroni correction, we were able to observe a significantcorrelation between tS6RP and tVEGFR2 (P < 0.0001) in theBev–Ev study. Furthermore, significant correlations were alsonoted between pEGFR and pS6RP (P ¼ 0.0018) and tEGFR andtVEGFR2 (P ¼ 0.0072) in the BEE study.

Average vascular score in dermal wound tissueAs a physiologic response to a wound, the dermis around the

punch biopsy undergoes an ordered series of vascular responses,which can be reproduciblymeasured (1).Wounds are imaged at aspecific time point and subsequently divided into 12 distinct

Figure 3.Changes in phospho VEGFR2 levelsfrombaseline. A–G, eachwaterfall plotrepresents a specific treatmentregimen in each trial. y-axis representsthe log fold change in phosphoVEGFR2 protein level from baseline.Each line along x-axis represents anindividual patient. H, percent changein phospho VEGFR2 protein levelsfrom baseline in response to thedifferent treatment regimens. Ev,everolimusmonotherapy; Erl, erlotinibmonotherapy; BE, bevacizumab þeverolimus double therapy; BEE,bevacizumabþ everolimusþerlotinibtriple therapy; BEP, bevacizumab þeverolimus þ panitumumab tripletherapy; BEE_BEP, BEE and BEPregimen combined; Pooled, alltreatment regimen combined.

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regions that are individually scored. The resulting AVS across allregions is then calculated (6). AVSwas assessed one week after thewound stimulus, just before the granulation tissue being har-vested for molecular analyses. AVS data from matched sets ofpretreatment and on-treatment images were available for 22patients on the BEE study, for 24 patients on the BEP study, and36 patients on the BE study.

Patients treated on the BE study exhibited AVS scores that weresignificantly decreased bybevacizumab treatment in combinationwith everolimus (P ¼ 0.009; Supplementary Fig. S1A). Of the 36evaluable patients, 25 patients had decreased AVS scores afterbevacizumab and everolimus. Patients treated on the BEP study

also exhibited AVS scores thatwere significantly decreased (18 outof 24 evaluable patients) by bevacizumab treatment in combi-nation with everolimus and panitumumab (P ¼ 0.023; Supple-mentary Fig. S1B). Although not statistically significant, a trendfor a decrease in AVS score after BEE triplet therapywas noted (P¼0.098; Supplementary Fig. S1C).

DiscussionFor essentially all novel targeted therapies, reliable pharmaco-

dynamic assays are critical for optimal dose selection and tounderstand the downstream consequences of target inhibition.

Figure 4.Changes in total S6RP levels frombaseline. A–G, each waterfall plotrepresents a specific treatmentregimen in each trial. y-axis representsthe log fold change in total S6RPprotein level from baseline. Each linealong x-axis represents an individualpatient. H, percent change in totalS6RP protein levels from baseline inresponse to the different treatmentregimens. Ev, everolimusmonotherapy; Erl, erlotinibmonotherapy; BE, bevacizumab þeverolimus double therapy; BEE,bevacizumabþ everolimusþ erlotinibtriple therapy; BEP, bevacizumab þeverolimus þ panitumumab tripletherapy; BEE_BEP, BEE and BEPregimen combined; Pooled, alltreatment regimen combined.






These assays need to be pragmatic, quantitative, and amenable toassessments at both pretreatment and on-treatment. These assaysshould also be fit for purpose and reflect the known biology of thetarget.

The current set of biomarker studies further demonstrates thefeasibility of using our dermal wound model. We confirmed thatour average vascular scoring analysis (AVS) provides semiquanti-fiable measures for overall vascularization of the wound and thatantiangiogenic agents inhibit this process. In this report, wedemonstrate that molecular analysis of granulation tissue canprovide a quantitative assessment of target inhibition formultiple

targeted therapies, provided the targets are readily expressed ingranulation tissue. Inhibition of VEGFR2 was demonstrated forbevacizumab, inhibition ofmTOR for everolimus, and inhibitionof EGFR for panitumumab. In this analysis, the model appears tobe sensitive enough to detect these effects in a limited number ofpatients and robust enough to demonstrate these effects acrossthree different trials with different targeted therapies. This modelwas also quantitative enough to detect potentially meaningfuldifferences in the levels of target inhibition.

These current biomarker studies are among the first to dem-onstrate direct target inhibition for a VEGF inhibitor. The

Figure 5.Changes in phospho S6RP levels frombaseline. A–G, each waterfall plotrepresents a specific treatmentregimen in each trial. y-axis representsthe log fold change in phospho S6RPprotein level from baseline. Each linealong x-axis represents an individualpatient. H, percent change in phosphoS6RP protein level from baseline inresponse to the different treatmentregimens. Ev, everolimusmonotherapy; Erl, erlotinibmonotherapy; BE, bevacizumab þeverolimus double therapy; BEE,bevacizumabþ everolimusþerlotinibtriple therapy; BEP, bevacizumab þeverolimus þ panitumumab tripletherapy; BEE_BEP, BEE and BEPregimen combined; Pooled, alltreatment regimen combined.


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inhibition of VEGFR2 phosphorylation by bevacizumab in gran-ulation tissue was consistently seen across our trials and acrosscohorts within trials. Furthermore, a reduction in total VEGFR2levels in granulation tissue was highly correlated with changesin vascularization in the dermis at the wound periphery. Theability to correlate molecular and physiologic changes in angio-genesis may be particularly important for the evaluation ofnovel antiangiogenesis agents and combination antiangiogenicregimens. These data also suggest that this model may be usefulto better understand the pathophysiology of these agents onwound healing in humans. Importantly, there were no signif-icant wound-healing complications from the biopsies done on

these studies and all wounds did heal, although many hadsignificant delays. Because cancers are known to co-opt manywound-healing programs (15, 16), these findings support thepotential relevance of our dermal wound model for studyingthe mechanisms of action, toxicity, and resistance for sometargeted therapies.

Previous biomarker studies with bevacizumab have notedchanges in vascular density (17) and vascular maturity (18), aswell as changes in phosphorylated VEGFR2 (19). These importantstudies, however, relied upon IHCmethods and needed to enrollselect patient populations. Our results are consistent with thosereports and themethods used here aremore readily adapted to the

Figure 6.Changes in total and phospho EGFRlevels from baseline. Each waterfallplot represents a specific treatmentregimen in each trial. y-axis representsthe log fold change in total EGFRprotein levels from baseline (A–D) orthe log fold change in phospho EGFRprotein levels from baseline (E–H).Each line along x-axis represents anindividual patient.






broader populations that comprise most dose finding phase Istudies.

Wewere also able to demonstrate target pathway inhibition forthe mTOR inhibitor everolimus. The mTOR pathway is complex,with multiple signaling nodes and feedback loops, includingHIF1a and IGFR (20). Although we were able to reliably analyzeS6RP, a known downstream effector ofmTOR,we did not analyzeadditional potential effectors due to the technical limitationsrelated to available reagents and the small size of granulationtissue samples. This effect on S6RP was consistent across differentstudies and cohorts, even though everolimus doses were signif-icantly reduced when combined with bevacizumab, panitumu-mab, or erlotinib. mTOR inhibitors have antiangiogenic proper-ties and have been associated with wound healing complications(21); the loss of total VEGFR2 on everolimus detected in ourmodel was consistent with a reduction in endothelial cell numberon treatment.

EGFR inhibition was observed in our analysis of the anti-EGFRantibody, panitumumab, but not the anti-EGFR tyrosine kinaseinhibitor, erlotinib. The doses of erlontinb and panitumumabused in most patients in our studies were 50% and 75% of theirtypical monotherapy doses, respectively. The lack of detectableEGFR inhibitionwith erlotinibmay reflect the lower doses used inthese studies. These differences may also reflect mechanisticdifferences between monoclonal antibody and TKI inhibition ofEGFR. The severity of skin rash has been noted to differ amongerlotinib, cetuximab, and panitumumb (22). Our data suggest,that these differences could be related to different levels of EGFRinhibition. Additional studies, particularly head to head compar-isons of monotherapy treatments, would be needed to confirmany differences between these agents or between different doseswith respect to target inhibition and downstream effects. Ourmodel provides a convenient and quantitative platform for suchcomparisons.

It should be pointed out that this report has several limitations.Our biomaker substudy analyses were implemented into thetreatment regimens and were explored based on the feasibilityof integrating this approach into traditional phase I and II testing.In this context, testing of all treatment monotherapies and com-binations was not possible. For this reason, we focused on thecohorts of greatest interest. Similarly, sample sizes were driven bythe clinical needs of the study and were thus based upon prag-matic clinical considerations, not formal power calculationsaround biomarkers. Nevertheless, our results were remarkablyconsistent across three separate studies. Finally, we did notperform simultaneous tumor biopsies in these studies to directlycompare findings in tumors and wounds. Although tumors havebeen compared with wounds that do not heal, all tumors are notthe same, nor are all wounds. This fact emphasizes the need forunderstanding the similarities and differences in tumor and

wound tissues and the need to apply this assay, like all surrogatebiomarker assays, in a fit for purpose framework. Nevertheless,our findings are consistent with numerous molecular compar-isons that have shownnumerous similarities between tumors andwounds (23–26).

In conclusion, our dermal wound model provides direct evi-dence for target inhibition of VEGFR2 by bevacizumab, mTOR byeverolimus, and EGFR by panitumumab. These results wereconsistent across several phase I and II studies. This dermalwoundmodel may also be useful to evaluate other novel targeted ther-apies and combinations.

Disclosure of Potential Conflicts of InterestH. Hurwitz is a consultant/advisory board member for Amgen, Genentech/

Roche, and Lilly/Imclone, and reports receiving commercial research grantsfrom Amgen, Bristol-Myers Squibb, Genentech/Roche, Lilly/Imclone, andNovartis. A. Nixon is a consultant/advisory boardmember for GlaxoSmithKlineand Novartis, and reports receiving commercial research grants from Amgen,Pfizer, and Tracon Pharmaceuticals. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: H. Hurwitz, A.B. NixonDevelopment of methodology: J. Jia, H. Hurwitz, A.B. NixonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Jia, E. Weiss, L. Howard, N. Kaplan, H. HurwitzAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Jia, A. Dellinger, A. Bulusu, E. Weiss, H. Pang,H. Hurwitz, A.B. NixonWriting, review, and/or revision of themanuscript: J. Jia, A.Dellinger, E.Weiss,C. Rushing, H. Pang, H. Hurwitz, A.B. NixonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Dellinger, E. Weiss, H. Li, N. Kaplan,H. HurwitzStudy supervision: H. Hurwitz, A.B. Nixon

AcknowledgmentsThe authors thank the patients and their families for their contributions to

our study, the Phase I research nursing staff and physicians for their tremendouseffort in carrying out these clinical trials, and the support of the Duke ClinicalResearch Unit and Duke Clinics, where the biopsies were performed. We alsoacknowledge the helpful discussions on this topic with Dr. Harold Dvorak.

Grant SupportThe parent clinical trials were supported by Genentech/Roche and Novartis.

This correlative work was supported with funding from the NIH (RO1-CA112252 and K24-CA113755).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 3, 2014; revised March 13, 2015; accepted April 1, 2015;published OnlineFirst April 15, 2015.

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Published OnlineFirst April 15, 2015.Clin Cancer Res Jingquan Jia, Andrew E. Dellinger, Eric S. Weiss, et al. and mTOR Therapies in a Clinical Model of Wound HealingDirect Evidence of Target Inhibition with Anti-VEGF, EGFR,

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