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Cancer Therapy: Clinical

BRAF Inhibition Is Associated with Enhanced MelanomaAntigen Expression and a More Favorable TumorMicroenvironment in Patients with Metastatic Melanoma

Dennie T. Frederick1, Adriano Piris3, Alexandria P. Cogdill1, Zachary A. Cooper1, Cecilia Lezcano6,Cristina R. Ferrone1, Devarati Mitra4, Andrea Boni1, Lindsay P. Newton1, Chengwen Liu7, Weiyi Peng7,Ryan J. Sullivan2, Donald P. Lawrence2, F. Stephen Hodi5, Willem W. Overwijk7, Gregory Liz�ee7,George F. Murphy6, Patrick Hwu7, Keith T. Flaherty2, David E. Fisher4, and Jennifer A. Wargo1

AbstractPurpose: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with

metastatic melanoma.

Experimental Design: Thirty-five biopsies were collected from 16 patients with metastatic melanoma

pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone

(vemurafenib) or BRAF þ MEK inhibition (dabrafenib þ trametinib) and were also taken at time of pro-

gression. Biopsieswere analyzed formelanoma antigens, T-cellmarkers, and immunomodulatory cytokines.

Results: Treatment with either BRAF inhibitor alone or BRAF þ MEK inhibitor was associated with an

increased expression of melanoma antigens and an increase in CD8þ T-cell infiltrate. This was also

associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase

in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the

immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression

and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with

combined BRAF and MEK inhibition.

Conclusions: Together, these data suggest that treatment with BRAF inhibition enhances melanoma

antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment,

providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly,

markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF

inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to

BRAF-targeted therapy in patients with melanoma. Clin Cancer Res; 19(5); 1225–31. �2013 AACR.

IntroductionMelanoma remains amajor world health problem (1, 2),

although recent advances in targeted therapy against onco-genic BRAF for melanoma have shown some promisingresults (3). Somatic mutations in the BRAF oncogene occur

in more than half of melanomas, with the vast majority ofthese harboring an activating point mutation (V600E;ref. 3). This oncogenic mutation leads to constitutive acti-vation of the mitogen-activated protein kinase (MAPK)signaling pathway and increased oncogenic potentialthrough a variety of mechanisms including reduced apo-ptosis, increased invasiveness, and increased metastaticbehavior (4). Recent in vitro data also suggest thatBRAFV600E could also contribute to immune escape (5).

Targeted therapy against oncogenic BRAF for metastaticmelanoma results in objective responses in the majority ofpatients whose tumors harbor BRAFV600E (6). Despite this,resistance to therapy remains a significant issue, with amedian duration of response between 6 and 7 months(6). There is a great deal of ongoing research to determinemechanisms of resistance and strategies to overcome resis-tance (7–9). Multiple distinct mechanisms of resistancehave already been identified in recent months (10–13).

Combination of BRAF-targeted therapy with other signaltransduction inhibitors has been proposed on the basis ofevidence that other pathways become activated upon

Authors' Affiliations: Divisions of 1Surgical Oncology, 2Medical Oncol-ogy, 3Dermatopathology, and 4Dermatology,MassachusettsGeneral Hos-pital; 5Department of Medical Oncology, Dana-Farber Cancer Institute;6Division of Dermatopathology, Department of Pathology, Brigham andWomen's Hospital, Boston, Massachusetts; and 7Department of Melano-ma Medical Oncology, Center for Cancer Immunology Research, TheUniversity of Texas M.D. Anderson Cancer Center, Houston, Texas

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

D.T. Frederick, A. Piris, and A.P. Cogdill contributed equally to this work.

Corresponding Author: Jennifer A. Wargo, Division of Surgical Oncology,Massachusetts General Hospital, 55 Fruit St, Boston, MA 02114. Phone:617-643-6195; Fax: 617-724-3895; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1630

�2013 American Association for Cancer Research.

ClinicalCancer

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on April 14, 2020. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 10, 2013; DOI: 10.1158/1078-0432.CCR-12-1630

http://clincancerres.aacrjournals.org/

emergence of resistance (14) and such clinical approachesare already underway. Another potential approach involvescombining BRAF-targeted therapy with immunotherapy.This strategy is supported by recently reported data showingthat treatment of melanoma cells with BRAF-targeted ther-apy results in increased expression of melanocyte differen-tiation antigens (MDA) and increased recognition by anti-gen-specific T cells (5). These results were corroborated intumor biopsies from patients with metastatic melanomareceiving BRAF-targeted therapy and CD8þ T-cell infiltratecorrelated with response to therapy (15, 16).

We sought to test the hypothesis that BRAF-targetedtherapy is associated with improved melanoma antigenexpression and an enhanced immune response in patientswith metastatic melanoma. We also assayed immunomod-ulatory cytokines and markers of T-cell cytotoxicity as wellas T-cell exhaustion markers and the immunosuppressiveligand PDL1 to gain insight into potential means to mod-ulate the immune response to BRAF inhibition.

Materials and MethodsPatient samples

PatientswithmetastaticmelanomacontainingBRAFV600E

mutation (confirmed by genotyping) were enrolled onclinical trials for treatment with a BRAF inhibitor (vemur-afenib) or combined BRAFþMEK inhibitor (dabrafenibþtrametinib; Supplementary Table S1) and were consentedfor tissue acquisition per Institutional Review Board (IRB)-approved protocol. Tumor biopsies were conducted pre-treatment (day 0), at 10 to 14 days on treatment, and/or attime of progression if applicable. Formalin-fixed tissue wasanalyzed to confirm that viable tumor was present viahematoxylin and eosin (H&E) staining. Additional tissue

was snap-frozen and stored in liquid nitrogen or was imme-diately processed for purification of RNA.

Purification of total RNASampleswere homogenized anddisrupted using amortar

andpestle followedbyuse of aQIAshredder. AQIAcubewasused to harvest RNA using the RNeasy Mini Protocol(Qiagen).

Quantitative PCRTotal RNA (250ng)was used as template, and Superscript

VILO cDNA Synthesis Kit (Invitrogen) was used to generatecDNA. Quantitative real-time PCR was carried out on anApplied Biosystems 7300 machine.

ImmunohistochemistryTumor biopsies were stained with primary antibodies for

MART-1 (Covance, SIG-38160–1000), HMB-45(gp100)(Leica, PA0027), CD4 (Leica, NCL-CD4–1F6), CD8 (Leica.PA0183), Perforin (Santa Cruz, sc-374346), TIM-3 (R&DSystems, AF2365), PDL1 (LS-Bio, LS-B3368), or GranzymeB (Abcam, ab4059) followed by a secondary antibody forhorseradish peroxidase and then 3,30-diaminobenzidine(DAB) or blue chromagen. Stained slides were interpretedby a dedicated dermatopathologist.

Counting of CD8þ T cellsCD8þ T-cell count was conducted on slides from pre-

treatment and on-treatment tumor biopsies in 4 adjacenthigh-power fields (HPF) in the areas of highest density ofCD8-positive cells. Only positive signals with clear lym-phocyte morphology were evaluated.

ImmunofluorescenceSections from formalin-fixed, paraffin-embedded (FFPE)

melanoma tumor biopsies were deparaffinized, rehydrated,and endogenous peroxidase activity was blocked in 3%hydrogen peroxide in water. After rinsing, heat-inducedantigen retrieval was conducted. Nonspecific binding wasblocked by 20% serum blocker and endogenous avidin þbiotin blocking system. Primary antibody (monoclonalantibody targetingMART-1) and fluorescein isothiocyanate(FITC)-conjugated secondary antibody were then applied.Images were acquired using a Nikon Eclipse-80i fluores-cence microscope.

StatisticsStatistics were conducted using GraphPad Prism or the R-

statistical package.

ResultsBRAF inhibition is associatedwith increasedmelanomaantigen expression in tumors of patients withmetastatic melanoma

Given prior preclinical findings that BRAF inhibitionleads to increased expression of melanoma antigens inmelanoma cell lines and fresh tumor digests in vitro, we

Translational RelevanceThis study analyzes immune responses in serial tumor

biopsies of patients with metastatic melanoma treatedwith BRAF-targeted therapy. Significantly, we show thatBRAF inhibition is associated with an increase in mel-anoma antigen expression and T-cell infiltrate and adecrease in immunosuppressive cytokines in tumors oftreated patients. An increase in markers of T-cell cyto-toxicity was also noted. Interestingly, both melanomaantigen expression and T-cell infiltrate were abrogated atdisease progression. Paradoxically, BRAF inhibition wasassociated with an increase in T-cell exhaustion markersTIM-3 and PD1 and the immunosuppressive ligandPDL1. Together, these data support the hypothesis thatcombined BRAF-targeted therapy and immunotherapymay improve responses in patients with metastatic mel-anoma. This work provides the basis for future researchstudies and clinical trials that will focus on understand-ing the complex interplay between the tumor, immunesystem, and the tumormicroenvironment in response totargeted therapy.

Frederick et al.

Clin Cancer Res; 19(5) March 1, 2013 Clinical Cancer Research1226




sought to validate these findings in patients with metastaticmelanoma undergoing treatment with a BRAF inhibitor(patients 1–5) or combined BRAF þ MEK inhibitor(patients 6–18; Supplementary Table S1). We observed asignificant increase in mRNA levels for melanoma antigensassayed after treatment with BRAF inhibitor (Fig. 1A).Melanoma antigen expression increased by 4.9-, 16.4-,13.3-, and 14.1-fold in MART, TYRP-1, TYRP-2, and GP100respectively. Of note, there was no statistically significantdifference inmelanomaantigen expression inbiopsies frompatients receiving a BRAF inhibitor alone versus combinedBRAF þ MEK inhibition (P > 0.1) with the exception ofTYRP-2 (P < 0.04) which was higher in those treated withcombined BRAF þ MEK inhibition (data not shown).Findings of increased MART-1 expression were validatedvia staining of MART-1 protein using both immunohis-tochemistry (Fig. 1B and Supplementary Table S2) andimmunofluorescence (Fig. 1C and Supplementary Fig. S1).

BRAF inhibition is associated with increased CD8þT-cell infiltrate in tumors of patients with metastaticmelanomaNext, we conducted studies to test the ability of BRAF

inhibition to augment T-cell infiltrate based on our pre-

clinical data showing that increased MDA expression isassociated with increased recognition by antigen-specificT cells. Tumor biopsies from patients with metastatic mel-anoma undergoing treatment with a BRAF inhibitor werestained byH&E and using immunohistochemistry for CD8-positive cells.We observed a significant increase inCD8þ T-cell infiltrate by immunohistochemistry in 10 of 11 patients(Fig. 2A–C). There was no difference in CD4þ T-cell infil-trate (data not shown).

BRAF inhibition is associated with decreasedimmunosuppressive cytokines and markers of T-cellcytotoxicity but increased T-cell exhaustion markersand PDL1 in tumors of patients with metastaticmelanoma

Next, we analyzed the tumors of treated patients forexpression of immunosuppressive cytokines, markers ofT-cell cytotoxicity, T-cell exhaustion markers, and theimmunosuppressive ligand PDL1 to determine the effectsof BRAF inhibition on the tumor microenvironment. BRAFinhibition was associated with a significant decrease in theexpression of immunosuppressive cytokines interleukin(IL)-6 and IL-8, whereas there was no significant changein TGF-b and IL-10 (Fig. 3A). A significant increase in

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Figure 1. BRAF inhibition is associatedwith increasedmelanoma antigen expression in tumors of patients withmetastatic melanoma. Tumors were harvestedandmRNA levels of gp100,MART-1, TYRP-1, and TYRP-2 in patientswithmetastaticmelanoma undergoing treatment with a selective inhibitor of BRAFV600E

were assayed. A, expression levels of the respective MDAs are shown as fold increase over pretreatment value and are plotted on a log scale in a box andwhiskers plot (n¼ 12). The bottom and top of the 25th and 75th percentile, respectively, for all patients, with the bar indicating the median value. The whiskersindicate the extremes with open circles representing data points greater than 1.5 times the interquartile range. Immunohistochemistry (B;�40magnification)and immunofluorescence (C) staining for themelanoma antigenMART-1 in pretreatment and on-treatment biopsies (patient 2) was conducted to confirm thatprotein expression correlated with mRNA expression. All microscopy was repeated at least 3 times with representative examples shown. P values are from a2-tailed Student t test with a m of 1, which corresponds to no change in mRNA levels on treatment. �, P � 0.05.

BRAF Inhibition Facilitates a More Favorable Tumor Microenvironment

www.aacrjournals.org Clin Cancer Res; 19(5) March 1, 2013 1227




expression was also observed in markers of T-cell cytotox-icity (perforin, granzyme B; Fig. 3B). Of note, T-cell exhaus-tion markers TIM-3 and PD1 were also significantlyincreased after BRAF inhibition (Fig. 3B). Expression of theimmunosuppressive ligand PDL1 was also significantlyincreased following treatment with a BRAF inhibitor (Fig.3C). These were validated using immunohistochemistry formarkers with available antibodies (Fig. 3D and E). Of note,there was no change in HLA expression (SupplementaryFig. S2).

Melanoma antigen expression and CD8þ T-cellinfiltrate are decreased at time of progression andrestored through MEK inhibition

Tumor biopsies from patients withmetastatic melanomaundergoing treatment with a BRAF inhibitor who pro-gressed on therapy were assayed for melanoma antigenexpression and CD8þ T-cell infiltrate as described previ-ously. At the time of progression, there was a significantdecrease in melanoma antigen expression (Fig. 4A andSupplementary Fig. S3) and CD8þ T-cell infiltrate. One ofthese patients was later enrolled on a combination trialincorporating combined BRAF þ MEK inhibition (dabra-fenib þ trametinib) after progressing on BRAF monother-apy. A tumor biopsy conducted after initiation of combinedBRAF þ MEK therapy showed restoration of melanomaantigen expression and CD8þ T-cell infiltrate (Fig. 4A–C).

DiscussionThe advent of therapywith BRAF inhibitors has produced

remarkable clinical success (6) and brought new hope forpatients with metastatic melanoma. However, the impres-sive response rates have been tempered by a short durationof response in the majority of patients (6). There is anintense effort underway to better understand mechanismsof resistance to BRAF-targeted therapy (7–9, 13), withseveral strategies proposed to counter this resistance. Com-bination with other targeted agents (14) has been proposedand clinical trials are currently underway. The addition offurther MAPK blockade via use of an MEK inhibitor with aBRAF inhibitor extends the median duration of responsefrom 5.6 to 9.5 months, although virtually all patientsprogress on therapywith very few complete responses noted(17).

Clinical studies have also shown striking successes forimmunotherapy approaches in melanoma (18–20). Adop-tive therapy, vaccines, immunomodulatory approaches,and immune checkpoint/tolerance blockades have allexhibited promising results, with ipilimumab now U.S.Food and Drug Administration (FDA)-approved on thebasis of positive phase III clinical trial results in advancedmelanoma (18) and other immune checkpoint inhibitorsin clinical trials.

There is growing evidence that BRAF-targeted therapymay synergize well with immunotherapy. Prior in vitro

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Figure 2. BRAF inhibition isassociated with increased CD8þT-cell infiltrate in tumors of patientswith metastatic melanoma.Tumors biopsied pretreatment andon-treatment were stained for H&Eand immunohistochemistry (IHC)was conducted for CD8þ T cellswith a representative patientshown (A). CD8þ T-cell countswere conducted in a blindedfashion by a dedicateddermatopathologist (B). AverageCD8þ T-cell counts are plotted foreach patient with error barsrepresenting the standarddeviation of four measurements.CD8þ counts from all patients(n¼ 11) are expressed in a box andwhiskers plot both pre- andon-treatment (C).

Frederick et al.





findings showed that pharmacologic manipulation of theMAPK pathway results in increased melanoma antigenexpression in melanoma cells and this increase was associ-atedwith enhanced sensitivity to antigen-specific T cells (5).More recently, our group and others have provided

supporting evidence for these in vitro findings, showing anincrease in tumor infiltrating lymphocytes in patients withmetastaticmelanoma treatedwith BRAF inhibitors (15, 16).An increase in immune infiltrate was seen in virtually allpatients although the response to treatment was variable,raising the question as to whether the increase in T-cellinfiltrate is directly related to MAPK pathway blockade or ifit is secondary to tumor necrosis. The results in Fig. 4showing restored T-cell infiltrate with combination BRAFþ MEK would suggest that the increased T-cell infiltrate ismore likely due to MAPK pathway inhibition, althoughfurther studies are necessary to better understand theresponse.This article provides strong in vivo support but more

importantly shows novel findings showing that BRAF inhi-bition is associated with increased melanoma antigenexpression, increased markers of T-cell cytotoxicity, anddecreased expression of immunosuppressive cytokines—allenhancing the tumor microenvironment. However, furthercharacterization of the immune infiltrate reveals that theinfiltrating T cells show an exhausted phenotype (withincreased TIM-3 and PD1) and increased expression of the

immunosuppressive ligand PDL1. These results suggest thatBRAF inhibition promotes T-cell infiltration and increasedmelanoma antigen expression; however, the immuneresponse may be limited due to an increase in exhaustionmarkers on T cells and an increase in PDL1. These findingsare novel and have important clinical implications andmayimply that successful combination therapy may requireimmune checkpoint blockade to enhance antitumorimmunity.

Another novel finding in these studies was the observa-tion that the increase in melanoma antigen expression andCD8þ T-cell infiltrate was abrogated at the time of progres-sion.While intriguing, these findings need tobe validated ina larger cohort of patients. The patterns suggest that re-activation of the MAPK pathway is responsible for suppres-sion of melanoma antigens and re-establishment of animmunosuppressive tumor microenvironment. Thishypothesis is further supported by our findings that subse-quent MAPK pathway inhibition using an MEK inhibitorcan restore melanoma antigen expression and promoteinfiltration of CD8þ T cells. It is unclear whether the useof a different BRAF inhibitor (dabrafenib) or the addition ofthe MEK inhibitor contributed to this phenomenon,although recent literature would suggest that it is morelikely to be the latter (21). Of note, in our in vitro studiesleading up to these clinical findings (5) observed a delete-rious effect of MEK inhibition on T lymphocytes which

Figure 3. BRAF inhibition isassociated with decreasedimmunosuppressive cytokines andmarkers of T-cell cytotoxicity butincreased T-cell exhaustion markersand PDL1 in tumors of patients withmetastatic melanoma. Tumors wereharvested and mRNA levels of IL-6,IL-8, IL-10, and TGF-b (A), perforin(n ¼ 11), GranzymeB (n ¼ 11), TIM-3(n ¼ 14) and PD1 (n ¼ 14; B), andPDL1 (n ¼ 11; C) in patients withmetastatic melanoma undergoingtreatment with a selective inhibitor ofBRAFV600E were assayed. Allpatients are expressed in a box andwhiskers plot. Open circles representdata points greater than 1.5 times theinterquartile range. P valuesindicated are from a 2-tailed Studentt test with a m of 1, which representsno change in mRNA value withrespect to the pretreatment value.�, P � 0.05. Immunohistochemistry(�40 magnification) for the perforin,GranzymeB, and TIM-3 (patient 6; D)and PDL1 (patient 12; E) inpretreatment and on-treatmentbiopsies was conducted to confirmthat protein expression correlatedwith mRNA expression. The dottedline ¼ tumor–stroma interface andthe inset is the isotype-specificcontrol.






raised the potential concern that MEK inhibition inpatients may alter T-cell function. However in the presentstudy, we saw no significant difference in T-cell infiltratein patients receiving BRAF inhibitor monotherapy versusBRAF inhibitor plus MEK inhibitor. Functional studies onthese T cells were not conducted in these patients; how-ever, these initial data about T-cell infiltrate might suggestthat MEK inhibition does not significantly impact T-cellfunction.

On the basis of the data presented herein, one couldspeculate that augmenting the immune response by pro-viding pro-immune cytokines (such as IL-2) or immunecheckpoint inhibitors (such as monoclonal antibodies tar-geting CTLA-4, PD1, or PDL1) will act synergistically withthe immune infiltrate elicitedbyBRAF inhibition inpatientswith metastatic melanoma. Interestingly, Hooijkaas andcolleagues showed that anti-CTLA-4 mAb in combinationwith BRAF inhibition did not enhance tumor growth con-trol in an induciblemurinemodel; however, thismodel alsoshowed a decrease in T-cell infiltrate after BRAF inhibitionwhich is contrary towhatwe see in patients (22). In amousemodel of BRAFV600E melanoma, Koya and colleaguesshowed improved antitumor activity, in vivo cytotoxic activ-ity, and intratumoral cytokine secretion by adoptively trans-

ferred cells in combination with a BRAF inhibitor (23).Trials combining BRAF-targeted therapy and immuno-therapy in patients are currently underway, although nodata is currently available about response rates, durationof response, or findings from correlative studies. Coupledwith these preclinical studies, our present findings rein-force the rationale for combined BRAF-targeted therapyand immunotherapy in the treatment of metastaticmelanoma.

Future studies to further evaluate the immune responseduring BRAF inhibition are still needed. A deeper under-stating of how these treatmentmodalities interact with eachother will allow for optimal design of clinical trials tomaximize response rates and duration of response inpatients with metastatic melanoma.

Disclosure of Potential Conflicts of InterestF.S. Hodi has served as a non-paid consultant to Genentech-Roche and

received clinical trial support from Genentech. K.T. Flaherty has served as aconsultant to GlaxoSmithKline and Roche/Genentech. No potential con-flicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design:A. Piris, A. Boni, P. Hwu, K.T. Flaherty, D.E. Fisher,J.A. Wargo

Figure 4. Melanoma antigenexpression and CD8þ T-cellinfiltrate are decreased at time ofprogression and restored throughMEK inhibition. A, tumors wereharvested at time of progressionand at time of treatment withcombined BRAF inhibition andMEK inhibition for patient 3. mRNAlevels of the melanoma antigensgp100, MART-1, TYRP-1, andTYRP-2 were assayed. B,immunohistochemistry (IHC) wasconducted for CD8þ T cells onpatient tumor samples. C, CD8þT cells were identified and countedby a dedicated pathologist.Average CD8þ T-cell counts areplottedwith error bars representingthe SD of 4 measurements.�, P � 0.05.

Frederick et al.





Developmentofmethodology:A. Piris, C. Liu,W.W.Overwijk, P.Hwu, J.A.WargoAcquisition of data (provided animals, acquired and managedpatients, provided facilities, etc.): D.T. Frederick, A. Piris, A.P. Cogdill,Z.A. Cooper, C. Lezcano, D. Mitra, A. Boni, C. Liu, W. Peng, R.J. Sullivan,D.P. Lawrence, G.F. Murphy, P. Hwu, K.T. Flaherty, D.E. Fisher, J.A.WargoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):D.T. Frederick, A. Piris, A.P. Cogdill, Z.A.Cooper, C.R. Ferrone, L.P. Newton, D.P. Lawrence, F.S. Hodi, G.F. Murphy,K.T. Flaherty, J.A. WargoWriting, review, and/or revision of the manuscript: D.T. Frederick, A.Piris, A.P. Cogdill, Z.A. Cooper, C. Lezcano, C.R. Ferrone, R.J. Sullivan, D.P.Lawrence, F.S. Hodi, W.W. Overwijk, G. Lizee, P. Hwu, K.T. Flaherty, J.A.WargoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D.T. Frederick, A.P. Cogdill, D.P.Lawrence, K.T. Flaherty, J.A. WargoStudy supervision: D.E. Fisher, J.A. Wargo

AcknowledgmentsThe authors thank the patients who participated in these trials.

Grant SupportThis work is supported byNIH grants 1K08CA160692–01A1 (J.A.Wargo)

and 1U54CA163125–01 (J.A.Wargo, K.T. Flaherty, and F.S.Hodi), theMGHVaccine and Immunotherapy Center (J.A. Wargo), and the Claflin Distin-guished Scholar Award (J.A.Wargo), and through the generous philanthrop-ic support of several families whose lives have been affected by melanoma.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 18, 2012; revised December 6, 2012; accepted January 7,2013; published OnlineFirst January 10, 2013.

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2013;19:1225-1231. Published OnlineFirst January 10, 2013.Clin Cancer Res Dennie T. Frederick, Adriano Piris, Alexandria P. Cogdill, et al. Patients with Metastatic MelanomaExpression and a More Favorable Tumor Microenvironment in BRAF Inhibition Is Associated with Enhanced Melanoma Antigen

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braf inhibition is associated with enhanced melanoma antigen … · we sought to test the...

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