Bevacizumab plus Ipilimumab in Patients with Metastatic ... · Research Article Bevacizumab plus Ipilimumab in Patients with Metastatic Melanoma F. Stephen Hodi1, Donald Lawrence4,

Research Article

Bevacizumab plus Ipilimumab in Patients with MetastaticMelanoma

F. Stephen Hodi1, Donald Lawrence4, Cecilia Lezcano10, Xinqi Wu1, Jun Zhou1, Tetsuro Sasada1,Wanyong Zeng1, Anita Giobbie-Hurder2, Michael B. Atkins11, Nageatte Ibrahim1, Philip Friedlander12,Keith T. Flaherty4, George F. Murphy5, Scott Rodig5, Elsa F. Velazquez7,9, Martin C. Mihm Jr5, Sara Russell6,Pamela J. DiPiro3, Jeffrey T. Yap3, Nikhil Ramaiya3, Annick D. Van den Abbeele3, Maria Gargano1, andDavid McDermott8

AbstractIpilimumab improves survival in advanced melanoma and can induce immune-mediated tumor vasculopathy.

Besides promoting angiogenesis, vascular endothelial growth factor (VEGF) suppresses dendritic cellmaturation andmodulates lymphocyte endothelial trafficking. This study investigated the combination of CTLA4 blockade withipilimumab and VEGF inhibition with bevacizumab. Patients withmetastatic melanoma were treated in four dosingcohorts of ipilimumab (3 or 10mg/kg) with four doses at 3-week intervals and then every 12 weeks, and bevacizumab(7.5 or 15 mg/kg) every 3 weeks. Forty-six patients were treated. Inflammatory events included giant cell arteritis(n ¼ 1), hepatitis (n ¼ 2), and uveitis (n ¼ 2). On-treatment tumor biopsies revealed activated vessel endotheliumwith extensive CD8þ and macrophage cell infiltration. Peripheral blood analyses demonstrated increases inCCR7þ/�/CD45ROþ cells and anti-galectin antibodies. Best overall response included 8 partial responses, 22instances of stable disease, and a disease-control rate of 67.4%. Median survival was 25.1 months. Bevacizumabinfluences changes in tumor vasculature and immune responses with ipilimumab administration. The combinationof bevacizumab and ipilimumab can be safely administered and reveals VEGF-A blockade influences on inflam-mation, lymphocyte trafficking, and immune regulation. These findings provide a basis for further investigating thedual roles of angiogenic factors in blood vessel formation and immune regulation, as well as future combinations ofantiangiogenesis agents and immune checkpoint blockade. Cancer Immunol Res; 2(7); 632–42. �2014 AACR.

IntroductionCTLA4 blockade with ipilimumab improves survival in

patients with metastatic melanoma when compared with agp100 peptide vaccine (1) and in combination with dacar-bazine chemotherapy when compared with dacarbazinealone (2). Efforts to further enhance the efficacy of immunecheckpoint blockade through rational treatment combina-tions are needed.

In pursuit of predictive markers, pretreatment levels ofvascular endothelial growth factor (VEGF-A) influence clinicaloutcomes to ipilimumab therapy (3). Therefore, determinants

that may limit ipilimumab efficacy include immunosuppres-sive angiogenic factors such as VEGF. VEGF has profoundeffects on immune regulatory cell function, specifically inhibit-ing dendritic cell maturation and antigen presentation (4, 5).Furthermore, there is increasing evidence for the role angio-genic factors play in influencing lymphocyte trafficking acrossendothelia into tumor deposits (6). Previous studies havedemonstrated the effects of ipilimumab on vessels feedingtumor deposits, resulting in an immune-mediated vasculopa-thy (7). As a result of CTLA4 blockade, granulocytes andlymphocytes infiltrate the endothelia, resulting in its destruc-tion and tumor necrosis. The clinical efficacy of targetingVEGF-A and its effects on pathologic angiogenesis have beenextensively studied with the use of bevacizumab (8–14), andthese findings suggest a role in counteracting the immuno-suppressive actions of VEGF. Given the effects on tumorvasculature witnessed in patients with melanoma being trea-ted with ipilimumab and the known activity of bevacizumab,we conducted a phase I study to investigate the potentialsynergies of this combination in patients with metastaticmelanoma.

Materials and MethodsStudy design and treatment

The protocol (Supplementary Appendix A) was approved bythe Dana-Farber/Harvard Cancer Center institutional review

Authors' Affiliations: Departments of 1Medical Oncology, 2Biostatistics,and 3Imaging, Dana-Farber Cancer Institute; 4Massachusetts GeneralHospital Cancer Center; Departments of 5Pathology and 6Surgery, Brig-ham and Women's Hospital; 7Tufts University; 8Beth Israel-DeaconessMedical Center, Boston; 9Miraca Life Sciences, Newton, Massachusetts;10University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania;11Lombardi Cancer Center Georgetown University, Washington, Districtof Columbia; and 12Mount Sinai Medical Center, New York, New York

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: F. Stephen Hodi, Dana-Farber Cancer Institute,450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-5053; Fax:617-582-7992; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0053

�2014 American Association for Cancer Research.

CancerImmunology

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board, and all patients provided signed informed consent.Patient eligibility included measureable unresectable stage IIIor stage IV melanoma, Eastern Cooperative Oncology Group(ECOG) performance status of 0 or 1, adequate end-organfunction. Exclusion criteria included central nervous system(CNS) metastases, prior treatment with ipilimumab or bev-acizumab, history of autoimmune disease, melanomainvolvement in the gastrointestinal tract, ulcerated skinlesions, or ongoing treatment with full-dose warfarin, hep-arin equivalent, nonsteroidal anti-inflammatory drugs,aspirin within 10 days of enrollment, or any medicationthat inhibits platelet function within 2 weeks before enroll-ment. Four cohorts of patients each received four doses ofipilimumab at 3-week intervals and then every 3 months,plus bevacizumab every 3 weeks (continuous): cohort 1, ipi-limumab 10 mg/kg þ bevacizumab 7.5 mg/kg; cohort 2,ipilimumab 10 mg/kg þ bevacizumab 15 mg/kg; cohort3, ipilimumab 3 mg/kg þ bevacizumab 7.5 mg/kg; cohort4, ipilimumab 3 mg/kg þ bevacizumab 15 mg/kg (Supple-mentary Fig. S1). Cohorts that included 3 mg/kg were addedfollowing the approval of ipilimumab at this dose to gainsafety data and experience.Patients were first enrolled in cohorts of five with 10 mg/kg

of ipilimumab. The dose-limiting toxicity (DLT) period was 12weeks. If�3 of 5 patients in cohort 1 did not experience a DLT,then the study was permitted to proceed to the next cohort. If�3 patients in cohort 3 experienced a DLT, the study wasdesigned to stop. To ensure that toxicity at the maximumtolerated dose (MTD) was acceptable and to gain additionalexperience with this combination, an additional 12 patientswere accrued at MTD. There was a 94% probability of doseescalation if the true rate of DLT was no more than 20%. If thetrue rate of DLT exceeded 50%, the probability of escalationwas less than 50%.

Statistical analysisBest overall response (BORR) was defined as the propor-

tion of patients with complete or partial response at anytime while on study. The disease-control rate (DCR) was theproportion of patients with complete response, partialresponse, or stable disease. Differences in response rates orDCR by cohort were assessed using the Fisher exact test.Time to progression (TTP), overall survival (OS), and dura-tion of response were assessed using the method of Kaplan–Meier, with pointwise, 95% confidence intervals (CI) esti-mated using log[�log(survival)] methodology. Equality ofsurvival curves by cohort was assessed using the log-ranktest. Comparisons of the incidences of adverse events bycohort according to CTCAE 3.0 System Class were con-ducted using the Fisher exact test. All P values were two-sided, with statistical significance defined as P < 0.05. Therewere no corrections for multiple comparisons.

Flow cytometryPeripheral blood samples from patients receiving ipilimu-

mab alone on an expanded access study were obtained onDana-Farber/Harvard Cancer Center review board–approvedprotocols and used for comparison with samples from the

current trial. Peripheral bloodmononuclear cells (PBMC) wereisolated by Ficoll-Hypaque (GE Biosciences) density gradientcentrifuge. Cells were stained with fluorescent conjugatedantibodies, and analyzed on a FC500 FACS analyzer (BeckmanCoulter). A total of 1� 105 events were collected. FACS analysisresults are expressed as percentages (CD4-PE Cy7, CD8-PE Cy7, CD28-PerCP, and CD45RO-ECD antibodies werefrom Beckman Coulter, CCR7-FITC from R&D Systems,and CD57-PE from Abcam). Percentage increase (cutoff�5%) between pretreatment and posttreatment by 50% wasconsidered a significant increase. Differences in the propor-tions increased between ipilimumab and ipilimumab plusbevacizumab groups were determined by the Fisher exacttest.

ImmunohistochemistryMetastatic melanoma samples from patients that received

ipilimumab plus bevacizumab (n ¼ 10) and ipilimumab alone(n¼ 6) on an expanded access study were obtained before andafter treatment initiation. Ipilimumab-alone samples wereobtained from patients being treated on approved clinicaltrials. Posttreatment samples from both the current ipilimu-mab–bevacizumab trial and ipilimumab alone were obtainedat the same time from the initiation of therapy, immediatelyfollowing the completion of ipilimumab induction at approx-imately week 12.

Biopsies of end-organs suspected of inflammatory eventsrelated to treatment were obtained whenever possible. For-malin-fixed paraffin-embedded tissue sections were stainedfor biomarkers that included CD3 (Dako; A0452; 1:250), CD8(Dako; M7103; 1:100), CD20 (Dako; N1502; undiluted), Foxp3(BioLegend; 320102; 1:50), and CD163 (Vector Laboratories;VP-C374; 1:500) to characterize immune infiltrates. To assessvessel morphology and activation, CD31 (Dako; N1596; undi-luted) and E-selectin (Neuromics; MO20039; 1:50) antibodieswere used.

Humoral immune responses detected followingtreatment

Antibodies presented in posttreatment sera werescreened using ProtoArray Human Protein Microarray v5(Invitrogen) according to the manufacturer's instructions.Antibody targets were identified by a Z factor of �0.4. Thepresence of galectin antibodies in the sera were confirmedby immunoblot analysis using recombinant human galec-tins-1, -3, and -9 (R&D Systems). To compare antibodylevels as a function of treatment, galectin-1, -3, and -9immunoblots (blocking 5% BSA) were incubated withpretreatment and posttreatment sera (1:2,000 dilution inPBS with 2% BSA) overnight, followed by horseradishperoxidase–conjugated goat anti-human IgG antibody(Invitrogen), and visualized with enhanced chemilumines-cence. Densities of protein bands and backgrounds werequantified using NIH ImageJ software. After backgroundsubtraction, galectin antibody responses to treatment weredetermined by the formula: fold change ¼ (densitypost �densitypre)/densityPre. A fold change of �0.5 was considereda significant increase.

Bevacizumab plus Ipilimumab

www.aacrjournals.org Cancer Immunol Res; 2(7) July 2014 633




Figure 1. High-grade treatment-related adverse events. A, grade4 events. ALT, alanineaminitransferase (SGPT); AST,aspartate aminotransferase(SGOT). B, grade 3 events. Elevenstudy patients had treatment-related, grade 3 events [23.9%,(95% exact CI, 13%–39%)].Hematoxylin and eosin–stained,formalin-fixed, paraffin-embeddedtissue sections showing temporalartery, cut in cross section, withtransmural acute and chronicinflammation at�200 (C) and�400(D) finalmagnification. E and F, skinwith chronic inflammationintermixed with eosinophils withinthe mid-dermis at �400 (E) and�1,000 (F) final magnification.G and H, core of liver with acuteand chronic inflammation includingprominent eosinophils, at�400 (G)and �1,000 (H) final magnification.

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ResultsPatients and treatmentA total of 46 patients were treated (Fig. 1, CONSORT

diagram). Five patients in cohort 1 were first treated withoutany DLTs. Five patients were then treated in cohort 2 withouttoxicities; therefore, cohort 2 (ipilimumab 10 mg/kg plusbevacizumab 15 mg/kg) was determined to be MTD. Anadditional 12 patients were treated at MTD. The study wasamended to enroll 12 patients each to cohorts 3 and 4 followingthe regulatory approval of ipilimumab at 3 mg/kg. Demo-graphics, disease status, and prior treatment according tocohort are summarized in Supplementary Table S1. Patientswere predominantly male (61%) with a median age of 58 years(range, 25–80). Eighty-nine percent of the patients had anECOG performance status of 0. Forty-one patients (89%) hadstage IV melanoma and 5 (11%) had unresectable, stage IIIdisease. Thirteen (28%) patients had prior chemotherapy andradiation, 7 (15%) had radiation and no chemotherapy, 16(35%) had chemotherapy and no radiation, and 8 (17%) hadneither radiation nor chemotherapy. The median number ofsites of diseasewas 3 (range, 1–8): 83%had lymph node disease,63% had lung, and 61% had soft tissue. The median follow-upbased on patients alive at the time of data retrieval was 11.8months (range, 2.6–42.5).

Adverse eventsToxicities (regardless of attribution) for the first 12 weeks

representing the dose-limiting window are presented in Sup-plementary Table S2. Two patients experienced DLTs, 1from cohort 3 and 1 from cohort 4. No DLTs occurred incohort 1 or 2. There were no treatment-related deaths. All46 patients reported adverse events. The most commonlyreported adverse events (any grade) were fatigue (n ¼ 14),rash/desquamation (n ¼ 15), headache (n ¼ 16), and cough(n ¼ 17). Supplementary Table S3 presents the grade 3 andgrade 4 events for each dose level for all events and those eventspossibly, probably, or definitively related.High-grade (grade 3/4) treatment-related adverse events for

any time on study are summarized in Fig. 1. Grade 4 eventsincluded proteinuria and hepatic toxicities. Thirteen patientsexperienced high-grade adverse events. High-grade inflamma-

tory events revealed by posttreatment biopsies included giantcell (temporal) arteritis (Fig. 1C and D), dermatitis (palpablepurpura; Fig. 1E and F), and eosinophilic hepatitis (Fig. 1G andH). There was one on-treatment death due to disease progres-sion reported for a patient in cohort 3. The incidence of eventsper patient is summarized in Table 1.

Study outcomesTreatment with ipilimumab plus bevacizumab resulted in

morphologic changes in intratumoral endothelia withrounded and columnar CD31þ cells compared with pretreat-ment or posttreatment samples from patients receivingipilimumab alone (Fig. 2A and Supplementary Fig. S2). Toillustrate variations in pathologic responses observed, exam-ples of intermediate and no response are provided in Sup-plementary Fig. S3. Increased expression of E-selectin as afunction of therapy was observed with combined treatmentrelative to ipilimumab alone, revealing further biochemicalevidence for endothelial activation by the addition of bev-acizumab. Concentrated CD31 staining was observed at theinterendothelial junctions (Supplementary Fig. S2). Pathol-ogy review further revealed that these endothelial changeswere associated with extensive immune cell infiltration oftumors. Vessel density was not affected significantly bybevacizumab therapy.

With pathologic examination of immune infiltrates asso-ciated with treatment, significant trafficking of CD8þ T cellsand CD163þ dendritic macrophages (Fig. 3) across thetumor vasculature was witnessed in ipilimumab plus bev-acizumab posttreatment biopsies that was qualitativelyincreased in comparison with that elicited by ipilimumabalone. There was minimal change in FoxP3þ cellularcomposition.

We next sought to identify altered immune responses result-ing from bevacizumab plus ipilimumab combination therapy.To pursue functional changes, flow cytometry detecting T-cellphenotypes in the peripheral blood demonstrated enhance-ment in CCR7þ/�CD45ROþ populations for CD4þ (Fig. 4A) andCD8þ (Fig. 4B) T cells (individual responses SupplementaryFig. S4). Bevacizumab plus ipilimumab significantly increas-ed circulating memory cell phenotypes compared with

Table 1. Number of treatment-related adverse events per patient

N Mean SD Min Median Max

Overall 13 1.4 0.7 1.0 1.0 3.0Cohort1 1 2.0 — 2.0 2.0 2.02 (MTD) 6 1.2 0.4 1.0 1.0 2.03 3 1.7 1.2 1.0 1.0 3.04 3 1.3 0.6 1.0 1.0 2.0

NOTE:Worst grade of reported adverse event/toxicity per patient. Thirteen patients had treatment-related, grade 3 or 4 events [28.3%,(95% exact CI: 16%–43%)]. For patients with treatment-related, grade 3 or 4 events, the table summarizes the number per patient.Adverse events classified as "not related" are included. Grades: 1, mild; 2, moderate; 3, severe; 4, life-threatening; 5, death.






ipilimumab alone (Fig. 4C). Furthermore, analyses with post-treatment sera using protein arrays identified galectin-1 andgalectin-3 in 2 of 4, and 3 of 4 patients, respectively (Z factor¼0.49 and 0.7 for galectin-1, and 0.58, 0.83, and 0.87 for galectin-3). Galectin-9 was not included in the protein array. Given itsbiologic significance in immune regulation, galectin-9 wasincluded in subsequent analyses. The presence of anti-galectinantibodies in sera was further confirmed by immunoblot usingrecombinant proteins (Fig. 4D). Patients who received ipili-mumab plus bevacizumab had a significantly higher number ofresponses to galectins-1, -3, and -9 than patients who receivedipilimumab alone (Fisher exact P ¼ 0.02; Fig. 4E).

EfficacyThe median follow-up at the time of this analysis was 17.3

months (95% CI, 11.1–30.2months). Thirty-nine (85%) patientsstopped treatment, and 7 patients remained on study. Ninepatients (23%) discontinued treatment due to toxicity, 29(74%) due to progressive disease, and 1 (3%) due to withdrawalof consent. Themedian number of cycles patients received was7 (range, 2–37).

The 12-week response for the entire treated patient popu-lation was 10.9% (95% exact CI, 4%–24%; highest cohort 2,17.6%). Clinical activity is shown in Fig. 5. BORRwas 19.6% [95%exact CI, 9%–34%; highest cohort 2, 29.4% (95% exact CI, 10%–56%); 1 complete response; Supplementary Table S4]. In addi-tion to examples of pseudoprogression (Fig. 6), delayed bestresponses (Supplementary Fig. S5) were observed. Response

kinetics for all patients are presented in Supplementary Fig.S6A and for cohort 2 (MTD) in Supplementary Fig. S6B. DCR forall patients was 67.4% [95% exact CI, 52%–81%; cohort 2, 76.5%(95% exact CI, 50%–93%)]. There were no statistical differencesin the response or DCR between cohorts. Most durableresponses achieved best response after months of therapy.One patient in cohort 2 (MTD) experienced 7 months of stabledisease before a partial response and subsequently had acomplete response beginning 17 months following the initia-tion of therapy. Eleven patients in cohorts 1 and 2 are alive,months after discontinuation of therapy. The median TTP was9.0 months [95% CI, 5.5–14.5 months; longest cohort 2, 14.5months (95% CI 3.8–¥); Supplementary Fig. S7]. There were nodifferences in progression-free survival (PFS) by cohort (log-rank P ¼ 0.32).

Radiographic evidence for early metabolic antitumorresponses without significant anatomic response was exem-plified with PET/CT imaging (Fig. 6A–D). Transientincreases in lesion size with decreased density before grad-ual decrease in size with subsequent imaging (Fig. 6E–H)were observed in a number of patients, consistent withpseudoprogression.

Thirty (65%) patients were alive at the time of data retrieval.Kaplan–Meier estimates for TTP and OS are shown in Sup-plementary Fig. S7. Median OS was 25.1 months [95% CI, 12.7–¥; cohort 2, 25.1 months (95% CI, 9.6–¥)]. There were nodifferences in OS by cohort (log-rank P ¼ 0.98). The Kaplan–Meier estimate of 1-year OS was 79% (95% CI, 62%–89%), and

Figure 2. Intratumoral blood vessel changes from treatment with bevacizumab plus ipilimumab in subcutaneous melanoma deposits. A, characterization oftumor-associated vasculature before and while on therapy. Endothelial cells lining small vessels within melanomas of ipilimumab plus bevacizumab–treatedpatientswere rounded andcolumnar as assessedbyhematoxylin and eosin (H&E) andCD31 staining, in contrastwith pretreatment samples andon-treatmentsamples from patients receiving ipilimumab alone (column to extreme right; v, vessel). Scale bar, 50 microns. Endothelial cells in tumor deposits ofpatients receiving ipilimumab plus bevacizumabwere also associated with increased expression of E-selectin, and adhesion and diapedesis of CD8þ T cells.Enlarged central panels highlight the focally occlusive appearance of this endothelial activation [top, H&E; bottom, CD31; inset, E-selectin)]. Basemembraneof vessels approximated by dotted lines.

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6-month TTP was 63% (95% CI, 47%–75%). The lower boundsof both confidence intervals exceed those of Korn andcolleagues (15).

DiscussionThe influences on immunologic effects witnessed with the

addition of bevacizumab to ipilimumab reveal novel mechan-isms of action for bevacizumab in patients. First, morphologicand biochemical alterations in the tumor vasculature resultedin endothelial activation associated with qualitative increasesin lymphocyte and myeloid/monocyte cell trafficking intotumor deposits. The monocytes had extensive dendritic pro-cesses. These patterns of immune infiltrates for some patientswere associated with transient increases in lesion size withdecreased density before gradual decrease in size with subse-quent imaging. Detailed characterization of these infiltratesand cellular functions are areas for future investigation. Vesselchanges were similar to those observed in high endothelialvenules (HEV) found in secondary lymphoid organs and asso-ciated with lymphocyte extravasation (16). Such endothelial

appearances correlated with the ability of lymphocytes tomigrate into tissues. E-selectin expression induced by bevaci-zumab facilitates lymphocyte adhesion and rolling (17). Inaddition, CD31 influences adhesive and signaling functions forvascular cellular extravasation (18, 19). Specifically, the con-centration of CD31 staining at the interendothelial junctions ofbevacizumab plus ipilimumab–treated specimens indicatesvessels adapted for efficient lymphocyte trafficking (20). Theseresults are consistent with previous observations of anti-VEGFtreatment increasing lymphocyte tumor infiltrates in adoptivetherapy models (21, 22).

Further evidence for immunologic changes by the com-bination was demonstrated in the peripheral blood throughincreasing circulating memory T cells resulting from theaddition of bevacizumab. This provides a definitive role forbevacizumab in effecting broad changes in the circulatingimmune composition. Furthermore, the coordinated T- andB-cell responses witnessed previously with checkpointblockade (23, 24) are confirmed by the increased antibodyrecognition of the galectin family members. This findingsuggests that the concerted effects of combination therapy

Figure 3. Histologic changes in tumor deposits resulting from treatment with bevacizumab plus ipilimumab. A, phenotypic characterization of immune-cell infiltrates in biopsies from responders before and after initiation of therapy. Tumors after initiation (ON) of ipilimumab–bevacizumab therapywere characterized as compared with pretreatment samples (PRE). Significant infiltration by CD3þCD8þ T cells and CD163þ macrophageswith minimal change in Foxp3þ component was observed. The enlarged panels (center) emphasize the tumor-infiltrating architecture of the immuneresponse (top left, skeletal muscle). In contrast, patients treated only with ipilimumab showed a lesser degree of immune-cell infiltrationwhile on therapy. The two ipilimumab–bevacizumab specimens are subcutaneous tissues, and the ipilimumab-alone specimen was from theoropharyngeal submucosa.






Figure 4. Cellular and humoral immuneresponses in the peripheral blood arealtered by the addition of bevacizumab toipilimumab. All samples were obtainedpretreatment and at week 12 at thecompletion of ipilimumab or ipilimumab–bevacizumab induction. A, example ofchanges as a function of treatment inCD4þCCR7þCD45ROþ and CD4þ

CCR7�CD45ROþ T-cell populations toipilimumab plus bevacizumab treatment,compared with changes with ipilimumabtreatment alone. B, example of changesas a function of treatment in CD8þ

CCR7þCD45ROþ and CD8þ

CCR7�CD45ROþ T-cell populations toipilimumab plus bevacizumab treatment,compared with the responses toipilimumab treatment alone. C, numberof patients with melanoma who have atleast 50% enhancement in CD4þ/CD8þ

CCR7þCD45ROþ and CD4þ/CD8þ

CCR7�CD45ROþ T-cell populationsfollowing treatment with ipilimumab(3 mg/kg), or ipilimumab (3 mg/kg) plusbevacizumab, or ipilimumab (10 mg/kg)plus bevacizumab. �, P < 0.05 betweenipilimumab and ipilimumab plusbevacizumab;

��,P<0.01.D, representative

immunoblots of antibody responses in 4bevacizumab plus ipilimumab–treatedpatients to a total of zero, one, two, or threegalectins. Arrows, increased antibodylevels in posttreatment sera samples.Galectin-1, -3, and -9 proteins were mixedtogether and equally loaded and separatedby gel electrophoresis. Following transferonto a membrane, strips were incubatedwith equally diluted pretreatment andposttreatment sera. Density analysis usingNIH ImageJ software confirmed theincreases in densities of the indicatedbands after subtracting background takenfrom nearby areas of each band. As inmostof the cases, density change was seen inonly one or two of the three galectins;protein(s) without density change served asloading controls. E, antibody responses togalectins in patients treated withbevacizumab plus ipilimumab andipilimumab alone. Anti-galectin-1,anti-galectin-3, and anti-galectin-9antibodies were detected in pretreatmentand posttreatment patient sera byimmunoblot analyses. Percentages ofpatients with increased levels of antibodiesto a total of zero, one, two, or threegalectins in bevacizumab plus ipilimumab-treated patients (n ¼ 45) and ipilimumab-alone patients (n ¼ 18). Density of eachband was measured using NIH ImageJsoftware and the antibody fold changewascalculated using the formula: fold change¼(densityPost � densityPre)/densityPre.Antibody levels were considered asincreased when the fold change � 0.5.

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can result in additional immune recognition that extendsimplications for both immune regulation and angiogenesis.As the galectin family members are involved in tumor-cellinvasion, metastases, and angiogenesis as well as immuneregulation, it will be important to define in future studies thefunctions of these antibodies and their potential therapeuticroles.In preclinical animal models and in humans, VEGF has

been associated with altered antitumor immune responses,including the suppression of dendritic-cell maturation (4, 5),proliferation of regulatory T cells, inhibition of T-cellresponses (25), and accumulation of myeloid-derived sup-pressor cells (26–28). In patients with colorectal cancer,bevacizumab improved the antigen-presenting capacityof circulating dendritic cells (29). The increase in dendrit-ic-cell infiltrates associated with the current study suggestsa role for VEGF blockade in influencing the associationof antigen-presenting cell with tumor cells and their mat-uration. This reveals an additional mechanism for bevaci-zumab on immune function in the context of checkpointblockade.Inflammatory toxicities were generally higher than

expected with ipilimumab alone, but remained manageable.These included rare vasculitic events suggesting immunerecognition of unique sets of antigens (30) and eosinophilic-driven processes. Importantly, there did not seem to bean increased incidence of dermatologic or gastrointestinalside effects such as colitis, which are most concerningfor ipilimumab. As such, the combination of blockingCTLA4 and VEGF-A may broaden the recognized antigenrepertoire.Recent clinical trials reporting survival outcomes for

patients with metastatic melanoma have led to regulatory

approval for ipilimumab and vemurafenib. Phase II trials ofipilimumab revealed 2-year survival rates of 24.2% to 32.8%(31, 32). In patients who had received at least one priortherapy, ipilimumab, when compared with a gp100 peptidevaccine, improved the median OS from 6.4 to 10.1 months(1). OS in previously treated patients with melanoma receiv-ing vemurafenib was 15.9 months (33). In the current phase Itrial combining bevacizumab and ipilimumab, the clinicalactivity was favorable compared with ipilimumab alone.As all but one partial response occurred in cohorts 1 and2 (10 mg/kg ipilimumab), the ipilimumab dose may influ-ence the efficacy when ipilimumab is combined with bev-acizumab. The median survival of the entire 46-patientcohort was greater than 2 years with significant antitumoractivity witnessed at MTD. With the lower bounds of bothconfidence intervals exceeding those of Korn and colleagues(15), this combination is worthy of further pursuit. Impor-tantly, these results establish a mechanistic foundation forcombining antiangiogenesis with immune checkpoint block-ade for cancer treatment.

Antiangiogenic therapy for cancer has traditionally pro-vided a means to limit the blood supply and improve deliveryof antineoplastic agents to tumor sites. When combinedwith chemotherapy or interferon, bevacizumab has provedefficacious at improving the outcomes of patients withcolorectal cancer, renal cell carcinoma, non–small cell lungcancer, breast cancer, ovarian cancer, and glioblastomamultiforme (8–12, 34, 35).

For many tumors, hypoxia creates a microenvironment ofinhibitory inflammatory cells (27, 36). The role of angiogenicfactors in suppressing inflammation to promote vessel growthhas increasingly been recognized in the context of tumorimmunology (36). The ability to counteract the dual function

Figure 5. Activity in treated patientsby cohort according to RECISTcriteria. Arrows, patients alive at thetime of analysis; crosses, death;black bars, discontinuation oftreatment other than due toprogressive disease. Five patientscame off trial due to toxicityrequiring systemic steroids. Onepatient withdrew consent afterweek 12 without DLT. CR,complete response; PD,progressive disease; PR, partialresponse; SD, stable disease.






of angiogenic factors in promoting vessel growth and suppres-sing immune responses is provided in the current study by theparallel effects on host immunity and tumor vasculature.Mechanisms defining the roles for bevacizumab in the contextof immune checkpoint blockade were uncovered. The combi-nation provoked inflammatory events in patients, promotedmemory cells circulating in the peripheral blood, enhancedintratumoral trafficking of effector cells, and improved endog-enous humoral immune responses to galectins. Further inves-tigation is needed to evaluate the mechanistic basis of bev-acizumab activity and the full impact of clinical activity.Continued development of immune checkpoint and antian-giogenic combination therapies are warranted for the treat-ment of melanoma and other cancers.

Disclosure of Potential Conflicts of InterestF.S. Hodi has received research support from Bristol-Myers Squibb

and Genentech, has an ownership interest (including patents) in IP licensed toBristol-Myers Squibb (as per institutional IP policy), and is a consultant/advisoryboard member for Bristol-Myers Squibb and Genentech. M.B. Atkins is aconsultant/advisory board member for Bristol-Myers Squibb and Genentech.E.F. Velazquez and M.C. Mihm serve as directors for SKADA, Inc., and areconsultant/advisory board members for Melasciences and Caliber ID.D. McDermott is a consultant/advisory board member for Bristol-Myers Squibb.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: F.S. Hodi, A. Giobbie-Hurder,Development of methodology: F.S. Hodi, W. Zeng, G.F. Murphy, J.T. YapAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F.S. Hodi, D. Lawrence, C. Lezcano, X. Wu, J. Zhou,T. Sasada, W. Zeng, M.B. Atkins, N. Ibrahim, P. Friedlander, K.T. Flaherty,

Figure 6. Example of pretreatment(A and B) and 8-weekposttreatment (C and D) PET CTimages. An early metabolicresponse is noted in adominant lefthepatic metastasis. E, axialcontrast-enhanced CT imageobtained at baseline demonstratesa solitary heterogeneoushypodense lesion in segmentII of the liver, consistent with ametastatic deposit. F, axialcontrast-enhanced CT obtained4 months after treatmentdemonstrates slight increase in thelesions with interval decrease indensity (from 40 HU to 19 HU,approximately 50%), consistentwith treatment effect. Theincrease in size representspseudoprogression. G and H,follow-up CT scans obtained 6 and36 months after the start oftreatment demonstrate gradualdecrease in size of the metastaticdeposit.


Hodi et al.




G.F. Murphy, E.F. Velazquez, M.C. Mihm, S. Russell, P. DiPiro, J.T. Yap, A.D. Vanden Abbeele, M. Gargano, D. McDermottAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F.S. Hodi, D. Lawrence, C. Lezcano, X. Wu, T. Sasada,W. Zeng, A. Giobbie-Hurder, P. Friedlander, K.T. Flaherty, G.F. Murphy, S. Rodig,E.F. Velazquez,M.C.Mihm, S. Russell, P. DiPiro, J.T. Yap, N. Ramaiya, A.D. Van denAbbeele, D. McDermottWriting, review, and/or revision of the manuscript: F.S. Hodi, D. Lawrence,C. Lezcano, X. Wu, A. Giobbie-Hurder, M.B. Atkins, N. Ibrahim, P. Friedlander, K.T. Flaherty, G.F. Murphy, S. Russell, P. DiPiro, J.T. Yap, N. Ramaiya, A.D. Van denAbbeele, M. Gargano, D. McDermottAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): F.S. Hodi, J. Zhou, W. Zeng, A.D. Vanden AbbeeleStudy supervision: F.S. Hodi

Grant SupportThis study was funded by NIH CA143832 (to F.S. Hodi) and

1R012CA158467 (to F.S. Hodi and G.F. Murphy), the Melanoma ResearchAlliance (to F.S. Hodi), the Sharon Crowley Martin Memorial Fund forMelanoma Research (to F.S. Hodi), and the Malcolm and Emily Mac NaughtFund for Melanoma Research (to F.S. Hodi) at the Dana-Farber CancerInstitute, Genentech/Roche, and Bristol-Myers Squibb.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 26, 2014; revised March 28, 2014; accepted April 7, 2014;published OnlineFirst April 21, 2014.

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Correction

Correction: Bevacizumab plus Ipilimumab inPatients with Metastatic Melanoma

In this article (Cancer Immunol Res 2014;2:632–42), which appeared in the July 2014issue of Cancer Immunology Research (1), the panels of Fig. 1 are labeled incorrectly.The grade 4 toxicities should be labeled as A, and the grade 3 toxicities as B; B toG should be labeled C to H. The authors regret this error.

Reference1. Hodi FS, Lawrence D, Lezcano C, Wu X, Zhou J, Sasada T, et al. Bevacizumab plus ipilimumab in

patients with metastatic melanoma. Cancer Immunol Res 2014;2:632–42.

Published OnlineFirst August 5, 2014.doi: 10.1158/2326-6066.CIR-14-0141�2014 American Association for Cancer Research.

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Bevacizumab plus Ipilimumab in Patients with Metastatic ... · Research Article Bevacizumab plus Ipilimumab in Patients with Metastatic Melanoma F. Stephen Hodi1, Donald Lawrence4,