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Research Article

Long-term Survival and Clinical Benefit fromAdoptive T-cell Transfer in Stage IV MelanomaPatients Is Determined by a Four-ParameterTumor Immune SignatureSara M. Melief1,Valeria V.Visconti1, Marten Visser1, Merel van Diepen2, Ellen H.W. Kapiteijn1,Joost H. van den Berg3, John B.A.G. Haanen3, Vincent T.H.B.M. Smit4, Jan Oosting5,Sjoerd H. van der Burg1, and Els M.E. Verdegaal1

Abstract

The presence of tumor-infiltrating immune cells is associatedwith longer survival and a better response to immunotherapy inearly-stage melanoma, but a comprehensive study of the in situimmune microenvironment in stage IV melanoma has not beenperformed. We investigated the combined influence of a series ofimmune factors on survival and response to adoptive cell transfer(ACT) in stage IV melanoma patients. Metastases of 73 stage IVmelanoma patients, 17 of which were treated with ACT, werestudied with respect to the number and functional phenotype oflymphocytes and myeloid cells as well as for expression ofgalectins-1, -3, and -9. Single factors associatedwithbetter survivalwere identified using Kaplan–Meier curves and multivariate Cox

regression analyses, and those factors were used for interac-tion analyses. The results were validated using The CancerGenome Atlas database. We identified four parameters thatwere associated with a better survival: CD8þ T cells, galectin-9þ dendritic cells (DC)/DC-like macrophages, a high M1/M2macrophage ratio, and the expression of galectin-3 by tumorcells. The presence of at least three of these parameters formedan independent positive prognostic factor for long-term sur-vival. Patients displaying this four-parameter signature werefound exclusively among patients responding to ACT andwere the ones with sustained clinical benefit. Cancer ImmunolRes; 5(2); 170–9. �2017 AACR.

IntroductionMelanoma is the most aggressive form of skin cancer and has

long been recognized as a highly immunogenic tumor and a goodtarget for immunotherapy (1). In different types of cancer, includ-ing melanoma, the presence of type I cytokine–oriented tumor-infiltrating lymphocytes (TIL) has been associated with improvedsurvival (2). Indeed, a strong ongoing immune response waslinked to spontaneous regression in about half of the primarymelanomas (3) and longer survival of patients with stage I–IIIprimary and regionally metastasized melanoma (4–6). Morerecently, a large study inpatientswith stage IV (distantmetastases)melanoma revealed that even at this stage, intratumoral T-cell

content was associated with improved survival (7). However, thepredictive value for survival was not so strong, indicating thatother immune-related factors previously studied in primary mel-anoma may also play a role (8–12); this involvement of otherimmune parameters was also suggested by studies at the geneexpression level (13, 14). In parallel, studies showing that a strongintratumoral T-cell infiltrate fosters a better response to PD-1checkpoint therapy (15) and autologous tumor cell vaccination(11), but also that intratumoralmacrophages can hamper CTLA-4checkpoint therapy (16), suggest that the tumor's immune con-texture may also influence the response to immunotherapy.

In this study, we have expanded on earlier studies (4, 7) byassessing the influence of a series of immune factors in themetastatic tumor microenvironment in a large group of stage IVmelanoma patients with up to 10 years of follow-up since metas-tasis. We identified four parameters, each of which was associatedwith better survival. These parameters comprised CD8 T cells, thepresence of galectin-9þ dendritic cells (DC)/DC-like macro-phages, a higher M1/M2macrophage ratio, and galectin-3 expres-sion by tumor cells. The presence of at least three parameters wasan independent prognostic factor for survival, which was validat-ed by analysis of these parameters in stage IV melanoma patientsin TheCancer GenomeAtlas (TCGA) database. Furthermore, withthe introduction of targeted therapies and checkpoint inhibitors,adoptive cell transfer (ACT) has mostly become a salvage therapy(17) for treatment of stage IV melanoma patients. Analysis of thepredictive value of this signature for the response to ACT revealedthat the pretreatment tumors of patients without clinical benefit

1Department of Clinical Oncology, Leiden University Medical Center, Leiden, theNetherlands. 2Department of Clinical Epidemiology, Leiden University MedicalCenter, Leiden, the Netherlands. 3Division of Immunology, The NetherlandsCancer Institute, Amsterdam, the Netherlands. 4Department of Pathology,Leiden University Medical Center, Leiden, the Netherlands. 5BioinformaticsCenter of Expertise, Leiden University Medical Center, Leiden, the Netherlands.

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

Corresponding Author: Els M.E. Verdegaal, Department of Clinical Oncology,Leiden University Medical Center, P.O. Box 9600, Leiden 2300 RC, the Nether-lands. Phone: 317-1526-3464; Fax: 317-1526-6760; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-16-0288

�2017 American Association for Cancer Research.

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(CB) predominantly display two or fewer of the beneficialimmune parameters, whereas the presence of three or four ofthese parameterswasmost frequent in patients showing sustainedCB after ACT.

Materials and MethodsPatient material

Formalin-fixed, paraffin-embedded tissue blocks from 73 stageIV metastatic melanoma patients undergoing surgery were col-lected at Leiden University Medical Center (LUMC, Leiden, theNetherlands) and at the Netherlands Cancer Institute (NCI,Amsterdam, the Netherlands). The patients were included inclinical studies that were approved by a local ethical committee(LUMC study P04.085, NCI study EudraCT 2010-021885-31),and all patients gave written informed consent. All specimenswere frommetastases, and biopsies were taken before any immu-notherapeutic treatment. Classification ofmetastaseswas done bytumor–node–metastasis (TNM) staging criteria (18), and infor-mation on the concentration of lactate dehydrogenase (LDH) atthe moment of sampling was collected. Included in the cohort of73patientswere 17patients thatwere treatedwithACT inongoingclinical studies in the LUMC and NCI. Of these patients, 7 wereclassified as patientswithoutCB [progressive disease (PD)] and10patients with CB (stable disease (SD), partial response (PR), andcomplete response (CR)] according to RECIST1.1 criteria.

Immunofluorescence and IHCThe presence of T-cell and macrophage infiltrate in the tumor

area and the expression of galectin-1, galectin-3, and galectin-9by the tumor was analyzed using previously determined optimalantibody concentrations and immunofluorescence staining pro-tocols as described before (19, 20). Briefly, T-cell infiltrates werestained with antibodies to CD3, CD8, and FoxP3. Macrophageswere identified using antibodies to CD14 and CD163. To exam-ine which cells expressed galectin-9, a small part of the cohortreceived triple immunofluorescence staining with antibodies togalectin-9, CD68, and CD11c. All secondary antibodies wereisotype-specific antibodies labeled with the fluorochromes AlexaFluor 488, 546, or 647. The expression of Tbet was analyzed byIHC as described before (19) with the exception that afterincubation with the primary Tbet antibody and incubation withBrightVision poly-HRP anti-mouse/rabbit/rat IgG, the antigen-antibody reactions were visualized using the NovaRED SubstrateKit for peroxidase (Vector Laboratories). For all antibody label-ing, negative controls and controls omitting the primary orsecondary antibody were performed. Positive control tissueslides were included for all antibody labeling, using tonsil forT cells, colon for galectin, and placenta for macrophage controls.Images were captured using a confocal microscope (LSM15,Zeiss) for the immunofluorescence labeling and a spectralmicro-scope (Leica DM4000 B, Leica Microsystems) for the immuno-histochemical stains. Random images (five per slide) were takenfor analysis. Analysis of the images was done using ImageJ.Intratumoral T cells, macrophages, galectin-9þ cells, and Tbetþ

cells were manually counted using the "cell counter" plugin ofImageJ and presented as number of cells/mm2 (average of fiveimages). Galectin-1 and galectin-3 expression by tumor cellswere analyzed using an immunoreactive score (IRS; ref. 21),taking into account the percentage of positive cells and theintensity of the staining (Supplementary Table S1A).

TCGA analysisFor validation of our results of the IHC and immunofluores-

cence experiments, we analyzed data from the publicly availableTCGA database (5). To reconstruct our parameters, we used geneexpression profiles of the subset of patients with stage IV mela-noma. CD8þ T cells were identified by taking the average of CD8Aand CD8B expression, galectin-9þ DC-like macrophages byGALS9 expression,M1/M2macrophage ratio by the ratio of CD86and CD163 expression, and expression of galectin-3 by tumorcells by LGALS3 expression. This approach assumes that the geneswe used are expressed preferably by the target cells for theparameter, and not in the other cell types in the sample. We usedthe z-scores as available in the TCGA data. This will yield a morecomparable weight of all parameters. For each parameter, a highand a low group was defined by splitting the samples on themedian value for the parameter.

Statistical analysisThe differences between different patient groups were ana-

lyzed using the nonparametric Mann–Whitney U test for com-parison of continuous variables between two groups and theone-way ANOVA or Kruskal–Wallis test for the comparison ofthree groups. For comparison of categorical data, thec2 test or theFisher exact test was used. Correlation analysis was done usingSpearman r correlation. Correlation between immune para-meters and overall survival (OS) since metastasis was calculatedby the Kaplan–Meier method and statistically analyzed by thelog-rank test. Univariate and multivariate Cox proportionalhazards models were used to determine the HR that representsthe relative risk of death among patients in the different indicatedgroups. In the multivariate Cox regression models, analysis wascorrected for age, serum LDH, and the pattern of visceral metas-tases (TNM staging), the latter two being established prognosticfactors for stage IV melanoma. Interaction analyses were per-formed on the parameters that were identified as prognostic forsurvival. For all tests, P values <0.05 were considered statisticallysignificant. For statistical analysis, the software package SPSSstatistics 20.0.0 was used (SPSS Inc.).

ResultsPatient characteristics

To study the immune markers that relate to survival in stage IVmetastaticmelanoma, tumor biopsies from a group of 73 patientswere investigated (Supplementary Table S1B). The mean age atthe moment of sampling was 52 (range, 25–74; 41 males and 32females). The patients were divided in tertiles (<9 months, 9–20months, >20 months) based on the survival of stage IV disease.The mean age of the group of patients with the longest survivalcohortwas a bit lower, but this differencewas not significant (one-way ANOVA, P ¼ 0.09). Maximal follow-up of the patients sincecollection of the metastatic sample was 120 months, and themedian survival since metastasis was 13 months (range, 1–120).Within the cohort of 73 patients, 17 patients were included thatwere treated with ACT. Of these 17 patients, 6 patients receivedT cells thatwere generated bymixed lymphocyte tumor cultures ofautologous tumor cells and peripheral blood mononuclear cells(22). The other 11 patients received T cells that were generated byrapid expansion of TILs (23). Within the ACT-treated group of17 patients, 10 were classified as having CB (3� SD, 4� PR, and3� CR). The CB patients showed a significantly better survival

A Beneficial Tumor Immune Signature in Stage IV Melanoma

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Figure 1.

Cell counts of immune infiltrate andexpression of galectin-1 and galectin-3 in theshort-, medium-, and long-term survivalcohorts. A, Cell counts (cells/mm2) and themedian are depicted from the number ofCD8þ T cells, CD4þ T cells, FoxP3þ T cells,and Total T cells (Tc) that infiltrated thetumor. B, Cell counts and median from theintratumoral M1 (CD14þCD163�) and M2(CD14þCD163þ) macrophages as well as theratio of M1/M2. C, Cell counts (cells/mm2) forgalectin-9þ cells and the IRS of galectin-1 andgalectin-3 on the tumor cells is shown.Statistical analysis of the differencesbetween the three patient groups wasperformed with the nonparametricKruskal–Wallis test.

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Figure 2.

CD8, galectin-3, galectin-9, and the M1/M2 ratio are associated with a longer survival. A, Kaplan–Meier curves showing the cumulative survival sincemetastasis for the patients with high and low number of CD8þ T cells, high and low ratios of M1/M2 macrophages, high and low numbers of galectin-9þ

(Gal9) cells, and high and low galectin-3 (Gal3) expression. B, Kaplan–Meier curves for the patient groups that show a combination of the indicated twoparameters as depicted in the graphs compared with all other patient groups. The depicted P values are from the log-rank test.






compared with the patients with no CB when calculated by theKaplan–Meier method and analyzed by the log-rank test (PD; P <0.001; Supplementary Fig. S1). The different methods used togenerate T-cell batches for ACT did not influence clinical outcome(Fisher exact test, P ¼ 0.64).

T cells, macrophages, galectin-3, and galectin-9 are prognosticfactors for survival

To study the immune signature in each tumor, the numbers ofT cells, macrophages, and galectin-9–positive immune cells werequantified per square millimeter of tumor area, whereas theexpression of galectin-1 and galectin-3 by tumor cells was deter-

mined via the IRS (Fig. 1; Supplementary Table S2A). CD8þ T cellswere defined as CD3þCD8þFoxP3–, CD4þ T cells were defined asCD3þCD8�FoxP3–, and regulatory T cells (Treg) were defined asCD3þCD8�FoxP3þ (Supplementary Fig. S2A). Two types ofmacrophages were defined: CD14þCD163– (M1) andCD14þCD163þ (M2) macrophages (Fig. 2B). Galectin-9 was,based on morphology, expressed by cells of myeloid origin(Fig. 2C). Additional costaining with CD68 and CD11c revealedthat a majority (70%) of these galectin-9–expressing cells coex-pressed CD11c, and part of these cells also expressed CD68,indicating that the galectin-9þ myeloid cells were predominantlyDCs or DC-like macrophages (Fig. 3A and B).

Figure 3.

Immune signatures of patients with short-, medium-, and long-term survival. A, The fraction of tumors displaying the number of positive immune parameters(0/4, 1/4, 2/4, 3/4, or 4/4) was plotted for the three survival groups. The fraction of tumors displaying a certain immune signature differed significantly between thethree survival cohorts (c2 test, P < 0.001). B, The correlation between the immune signature and survival was analyzed by the Kaplan–Meier method. C, Thecorrelation between immune signature was analyzed in an independent patient cohort of stage IV melanoma patients from the TCGA database by theKaplan–Meier method.

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Galectin-1 and galectin-3were expressed by the tumor cells, butwith different intensities between samples (Fig. 2C). Todeterminethe type I orientation of the TIL, part of the cohort was stained forTbet (Fig. 3C). A strong positive correlation between the numberof T cells and the number of Tbetþ cells was observed (Spearman r

correlation coefficient 0.720, P < 0.001; Supplementary Table S2),suggesting a type I immune contexture in strongly T-cell–infil-trated tumors (24). In addition, the number of galectin-9þ

DCs/DC-like macrophages was also strongly related to that ofintratumoral T cells and Tbetþ cells (Supplementary Table S2B),fitting with the notion that galectin-9 is expressed on immunecells upon exposure to proinflammatory mediators (25). Nocorrelation was found between OS and any of the investigatedmarkers (Supplementary Table S2B).

Quantification of the T cells revealed huge variability in thenumber of CD8, CD4, and Treg cells, as well as tumor-infiltratingM1 and M2 macrophages, within each survival group. Patientswith high T-cell counts and a higher ratio ofM1/M2macrophageswere more often found in the long- and medium-term survivalgroups (Fig. 1A and B). The number of patients with densegalectin-9þ DCs/DC-like macrophages was higher in the groupsof patients with medium- and long-term survival (Fig. 1C). Theexpression of galectin-1 and -3 by tumor cells was not so differentbetween the patient groups, albeit that in the long-term survivalgroup, galectin-1 expression was somewhat lower and galectin-3higher (Fig. 1C). Notably, none of the observed differences werestatistically significant between the three groups (SupplementaryTable S2).

To investigate the potential influence of all these parameters onsurvival since metastasis, a univariate and multivariate Cox anal-ysis was performed, and this revealed that high numbers of CD8þ

T cells and galectin-9þDCs/DC-like macrophages, a high M1/M2

ratio, and tumors expressing galectin-3were prognostic factors forsurvival (Table 1). For calculation of the correlation between theseparameters and survival using the Kaplan–Meier method, thepatients were stratified on the basis of the median cell counts orIRS. The presence of these four parameters was also related in thisanalysis to longer survival (Fig. 2A). Because these four differentelements in the tumor immune microenvironment were prog-nostic for survival, we performed interaction analyses of eachcombination of two parameters. Patients with a combination oftwoof anyof these parameters (CD8þhigh/M1/M2high/galectin-9þ high/galectin-3 high) showed better survival compared withthe other patients, resulting in decreased HRs in the multivariateCox regression analysis (Table 1) as well as higher survival rateswhen the Kaplan–Meier analysis was used (Fig. 2B). Each com-ponent positively contributed to the other (Fig. 4).

To investigate whether it would be possible to define a certainimmune signature for each of the three survival cohorts, thefraction of tumors displaying combinations of immune para-meters was plotted (Figs. 3A and 5). This revealed that thefraction of tumors displaying a certain immune signature dif-fered significantly between the three survival cohorts (c2 test,P < 0.001). The group with the longest survival contained thelargest fraction of patients with a tumor that was positive for fourbeneficial parameters, whereas this immune phenotype was notpresent in the short survival cohort. We then questioned howmany beneficial parameters were required to have a majorimpact on survival. The group of patients with metastases dis-playing two or less of the beneficial immune parameters showeda low survival rate. The survival rate increased when patientsdisplayed three or four of the immune parameters, with a 100%survival for patients with a tumor that displayed all four immuneparameters (Fig. 3B). In addition, amultivariate Cox analysis was

Table 1. Univariate and multivariate analysis of survival since metastasis

Variable Crude HR (95% CI) P Adjusted HRa (95% CI) P

Gender 0.867 (0.507–1.484) 0.603Age 1.027 (1.005–1.049) 0.017LDH level high 4.513 (2.080–9.793) 0.000TNM stage M1c 2.280 (1.275–4.075) 0.005

CD4þ infiltration 0.681 (0.340–1.160) 0.158 0.758 (0.421–1.364) 0.356CD8þ infiltration 0.561 (0.328–0.961) 0.035 0.583 (0.325–1.044) 0.058FoxP3þ infiltration 0.620 (0.362–1.062) 0.081 0.795 (0.443–1.428) 0.443Total T-cell infiltration 0.608 (0.356–1.037) 0.068 0.590 (0.334–1.043) 0.069Tbet 0.668 (0.378–1.181) 0.165 0.833 (0.446–1.557) 0.567CD8/Treg ratio 1.637 (0.956–2.801) 0.072 1.129 (0.631–2.020) 0.683Galectin-1 expression by tumor 1.245 (0.731–2.123) 0.420 1.461 (0.800–2.669) 0.217Galectin-3 expression by tumor 0.481 (0.275–0.839) 0.010 0.432 (0.239–0.782) 0.006Galectin-9 infiltration 0.575 (0.334–0.991) 0.046 0.712 (0.377–1.347) 0.297M1 infiltration 0.723 (0.425–1.230) 0.232 0.836 (0.460–1.521) 0.557M2 infiltration 1.307 (0.763–2.242) 0.330 1.374 (0.761–2.481) 0.293M1/M2 ratio 0.518 (0.297–0.903) 0.020 0.426 (0.227–0.802) 0.008

CD8 high & Gal3 high 0.316 (0.142–0.704) 0.005 0.278 (0.112–0.686) 0.005CD8 high & Gal9 high 0.468 (0.254–0.862) 0.015 0.600 (0.296–1.214) 0.156CD8 high & M1/M2 high 0.444 (0.203–0.867) 0.017 0.409 (0.191–0.876) 0.021Gal3 high & Gal9 high 0.331 (0.616–0.684) 0.003 0.324 (0.145–0.723) 0.006Gal3 high & M1/M2 high 0.339 (0.172–0.668) 0.002 0.289 (0.142–0.589) 0.001Gal9 high & M1/M2 high 0.357 (0.717.0.722) 0.004 0.434 (0.212–0.892) 0.023

3 or more parameters high 0.328 (0.177–0.608) 0.000 0.273 (0.134–0.555) 0.000

NOTE: Cox regression analyses. Crude HRs and adjusted HRs and the 95% confidence intervals for high versus low numbers of CD4 T cells, CD8 T cells, FoxP3 T cells,total T cells, and Tbetþ cells, high versus low CD8/Treg ratio, high versus low expression of galectin-1 and galectin-3 by the tumor, high versus low numbers ofgalectin-9þmyeloid cells, M1macrophages, andM2macrophages cells, and a highversus low ratio ofM1/M2macrophages sincemetastasis are shown. HRs for patientgroups showing a combination of markers versus all other patients are shown. Significant HRs are depicted in bold an italic numbers.aAdjusted for age, gender, LDH level, and TNM stage.






performed with age, gender, LDH level, and TNM stage ascovariates. This showed that an immune signature consisting ofmore than three parameters was an independent prognosticfactor for survival (Table 1).

To validate our results, this immune signaturewas analyzed in aset of stage IV melanoma patients in the TCGA database, whichcontained 23 patients. In this cohort, patients with a tumor thatdisplayed three or four of the immuneparameters hadbetter long-term survival (P < 0.001) compared with the rest of the patients(Fig. 3C).

More patients with a beneficial immune signature among thosewith CB from ACT

We analyzed 17 pre-ACT treatment biopsies of metastases.Their immune microenvironment showed that the tumors from

patients with CB showed higher numbers of intratumoral T cellscompared with the patients showing PD (Fig. 4; SupplementaryTable S2A). CB patients showed a significantly higher ratio ofM1/M2 macrophages, which was mostly the result of a low M2infiltration (Fig. 4). In addition, the number of intratumoralgalectin-9þ DCs/DC-like macrophages and tumors expressinggalectin-1 was lower in the CB patients. Next, the fraction oftumors displaying the combination of immune parameters linkedto better survival was plotted (Fig. 5A). Thirty percent of the CBpatients displayed an immune signature positive for four of ouridentified beneficial immune parameters, whereas none of the PDpatients showed this immune phenotype. Of the 5 CB patientsthat showed an immune signature of two or less beneficialimmune parameters, 4 showed a low infiltrate of CD8þ T cells(Supplementary Table S3), which potentially was corrected by theACT. Analysis of the effect of this immune signature on thesurvival after ACT revealed that sustainable CB was only foundin those patients whose tumor immune signature included threeto four of the identified parameters before treatment with ACT(Fig. 5B).

DiscussionIn this study, we investigated the tumor immune contexture in

stage IV metastatic melanoma and analyzed its association tosurvival. We found that an infiltrate of CD8þ T cells, galectin-9þ

DCs or DC-like macrophages, a high M1/M2 ratio, and a highexpression of galectin-3 by the tumor were associated with sur-vival. The groups of patients with an immune signature consistingof a combination of three or four immune contexture–definingelements displayed the longest survival. Notably, most of thepatients in our cohort were treated in the preimmunotherapy erawhen the OS generally was low (26). Since the introduction ofimmunotherapies, the OS of stage IV melanoma patients hasincreased considerably. Independent validationof our results on amore recent cohort of 23 stage IVmelanomapatientswith ahighermedian OS in the TCGA database (5) sustained our observationthat patients with tumors that expressed at least three of thedefined immune parameters survived longer than patients withtumors not displaying three to four of these immune parameters.Notably, the immune signature is also prognostic for longersurvival after ACT treatment and may be predictive for responseafter ACT, as treatment of stage IVmelanoma patients by adoptiveT-cell transfer was more likely to be successful and durable whenthe metastases of these patients displayed at least three of theidentified four beneficial immune parameters prior to treatment.However, a firm statement about the predictive value of theimmune signature requires validation in a prospectively ACT-treated patient cohort.

Our study clearly showed that not only T cells but multipleelements defining the tumor immune contexture are of impor-tance for the prognosis of stage IV metastatic melanoma patientsand their response to ACT. Rather than the numbers of severaltypes of infiltrating immune cells, which did not differ among theshort-, medium, and long-term survival groups, it is their com-bined presence and the processes they reflect that bears an impacton outcome. We showed that there were strong correlationsbetween the Tbetþ lymphocyte counts and CD4 and CD8 T-cellcounts, suggesting an ongoing type I cytokine intratumoral T-cellresponse. The notion of such a proinflammatory signature issustained by the presence of galectin-9þ myeloid cells, the

Figure 4.

Cell counts of immune infiltrate and expression of galectin-1 and galectin-3 inpatients that were treated with ACT. Cell counts (cells/mm2) and the medianare depicted of infiltrated immune cells [CD8þ T cells, CD4þ T cells,FoxP3þ T cells, total T cells, M1 (CD14þCD163�), and M2 (CD14þCD163þ)macrophages], the ratio of M1/M2, and expression (IRS) of galectin-1 andgalectin-3 on tumors of patients showing either PD or CB after treatment withACT. Differences between the two groups were statistically analyzed by aMann–Whitney U test (� , P < 0.05; �� , P < 0.01).

Melief et al.





number of which was strongly correlated to the number of Tbetþ

cells and T cells. Galectin-9 is known to be expressed by myeloidcells after exposure to proinflammatory cues in the microenvi-ronment (25, 27). On the basis of the coexpression of CD68 andCD11c, we conclude that these galectin-9þ cells predominantlyare DCs and DC-like macrophages that can be crucial for theinduction of strong antitumor immunity similar to their pub-lished role as crucial mediators of anticancer immune responsesafter anthracycline-based chemotherapy (28). It is not clearwhether galectin-9 is also secreted by these myeloid cells. If so,this could also contribute to the antitumor response, as galectin-9was shown to increase the numbers and cytolytic capacity of CD8T cells and natural killer (NK) cells in the Meth-A and B16F10mouse tumor models (29, 30). The presence and activity of NKcells was not assessed in our study but may form an additionalparameter associated with survival, as highly activated NK cellshave been found in tumor-infiltrating lymph nodes ofmelanomapatients (31). In addition, the presence of a proinflammatoryenvironment was reflected by the higher M1 to M2 macrophageratio found in long-term survivors. Previous studies inmelanomadid not find associations between macrophage infiltrate andsurvival, most likely because these studies did not distinguishbetween functionally differentmacrophages anddidnot relate thepresence ofmacrophages toother immune cells (4, 8, 32). Inothertypes of cancer, however, the presence of proinflammatory (M1)

macrophages and, in particular, a highM1 toM2 ratio was shownto be related to improved survival (33, 34).

In our study, a high expression of galectin-3 by the tumor wasassociated with long-term survival, which was also reported forprimary melanoma (12). This is counterintuitive as existingliterature mostly describes an immunosuppressive role for galec-tin-3 (35). Galectin-3 can hamper T-cell activation by interferingwith T-cell receptor signaling (36) and by binding to the check-point inhibitor LAG-3 (37). In addition, it has been shown toincrease apoptosis in effector T cells (38, 39). The positive effect ofgalectin-3 expression on survival is possibly explained by its directeffect on the tumor cell and might be a result of a highersusceptibility to oxidative damage and apoptotic death of galec-tin-3–expressing melanoma cells (40).

The presence of an at least three-parameter beneficial immunesignature was also found in the patients displaying sustained CBafter ACT. The other 5 CB patients, lacking this signature, dis-played only short-term benefit from ACT. None of the patientsthat were unresponsive to ACT showed an immune signaturewiththe presence of all four parameters. We hypothesize that thequality of the ACT T-cell product determines whether a patientresponds to therapy. Indeed, T-cell batches that were infused intoCB patients comprised tumor-specific T cells with predominantlya Th1 cytokineprofile,whereas the T-cell batches thatwere infusedinto the patientswith PD showed anon-Th1 cytokine profile (22).

Figure 5.

Immune signature of ACT-treatedpatients. A, The immune signatures forthe patients showing either PD (n ¼ 7)orCB (n¼ 10) after treatmentwithACT.Plotted are the fractions of patientsshowing tumors that were positive for0/4, 1/4, 2/4, 3/4, or 4/4 relevantimmune parameters. The type ofresponse is depicted for the patientsshowing CB. B, The correlationbetween the immune signature andsurvival was analyzed by the Kaplan–Meier method for the complete groupof ACT-treated patients. The patientgroup that was positive for 3 to 4 of 4parameters versus the group ofpatients that was positive for 0 to 2 of 4parameters was plotted. The depictedP value is from the log-rank test.






We speculate that even if good-quality ACT is given to patientswhose tumor displays a less desirable immune signature, inparticular those showing a failure to recruit large numbers of Tcells, only a short period ofCB canbe achieved. It canbe envisagedthat an initial failure to recruit T cells, potentially due to a lack of T-cell–attracting chemokines secreted by these tumors (13), willalso hamper ACT. Unfortunately, we do not have the tumormaterial after ACT treatment to test our hypothesis.

Our study is limited by the relatively low number of patientsthat we could analyze, in particular the patients treated withtumor-reactive T cells and the stage IV melanoma patients avail-able for analysis in the TCGA cohort. Validation of our results inother cohorts will be important to understand the true value ofour findings.

We conclude that a tumor immune contexture of at least threeof the four parameters described inour study is prognostic to long-term survival of stage IVmelanoma patients. Moreover, this sameimmune signature is required for sustainable responsiveness toACT of these patients. It would be of interest to validate thepredictive value of this immune signature in a prospective cohortof patients treated with ACT as well as to study whether thissignature may predict the response to immune checkpoint block-ers. If so, the use of this immune signature will be important forpersonalized therapy, that is, development of tailor-made therapywith higher predicted treatment benefit.

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

Authors' ContributionsConception and design: S.M. Melief, V.T.H.B.M. Smit, S.H. van der Burg,E.M.E. VerdegaalDevelopment of methodology: S.M. Melief, V.V. Visconti, E.M.E. VerdegaalAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.M. Melief, E.H.W. Kapiteijn, E.M.E. VerdegaalAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.M. Melief, M. Visser, M. van Diepen, J. Oosting,S.H. van der Burg, E.M.E. VerdegaalWriting, review, and/or revision of the manuscript: S.M. Melief, M. vanDiepen, E.H.W. Kapiteijn, J.H. van den Berg, J.B.A.G. Haanen, V.T.H.B.M. Smit,S.H. van der Burg, E.M.E. VerdegaalAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.M. Melief, M. Visser, J.H. van den BergStudy supervision: S.H. van der Burg, E.M.E. Verdegaal

AcknowledgmentsWe would like to acknowledge the NKI-AVL Core Facility Molecular Pathol-

ogy & Biobanking (CFMPB) for supplying NKI-AVL Biobank material and/orlaboratory support.

Grant SupportThis study was supported by the Landsteiner Foundation for Blood Trans-

fusion Research (project number 1207) and the Dutch Cancer Society (KWFUL2012-5544).

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 October 24, 2016; revised December 9, 2016; accepted December11, 2016; published OnlineFirst January 10, 2017.

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2017;5:170-179. Published OnlineFirst January 10, 2017.Cancer Immunol Res Sara M. Melief, Valeria V. Visconti, Marten Visser, et al. Four-Parameter Tumor Immune SignatureTransfer in Stage IV Melanoma Patients Is Determined by a Long-term Survival and Clinical Benefit from Adoptive T-cell

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