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Indoleamine 2,3-dioxygenase, a new prognostic markerin sentinel lymph nodes of melanoma patients

Reinhart Speeckaert a,*, Karim Vermaelen b, Nanja van Geel a, Philippe Autier c,Jo Lambert a, Marc Haspeslagh a, Mireille van Gele a, Kris Thielemans d, Bart Neyns e,Nathalie Roche f, Natacha Verbeke g, Philippe Deron h, Marijn Speeckaert g, Lieve Brochez a

a Department of Dermatology, Ghent University Hospital, Ghent, Belgiumb Department of Respiratory Medicine, Ghent University Hospital, Ghent, Belgiumc International Prevention Research Institute, Lyon, Franced Laboratory of Molecular and Cellular Therapy, Department of Immunology–Physiology,

Medical School of the Vrije Universiteit Brussel, Brussel, Belgiume Department of Medical Oncology, UZ-Brussel, Brussel, Belgiumf Department of Plastic and Reconstructive Surgery, Ghent University Hospital, Ghent, Belgiumg Department of Medical Oncology, Ghent University Hospital, Ghent, Belgiumh Department of Head and Neck Surgery, Ghent University Hospital, Ghent, Belgium

A R T I C L E I N F O

Article history:

Available online 25 October 2011

Keywords:

Indoleamine 2,3-dioxygenase

Melanoma

Prognosis

Regulatory T cell

Sentinel node

0959-8049/$ - see front matter � 2011 Elsevidoi:10.1016/j.ejca.2011.09.007

* Corresponding author: Address: Departmen+32 93322298; fax: +32 93324996.

E-mail address: Reinhart.Speeckaert@uge

A B S T R A C T

Background: Indoleamine 2,3-dioxygenase (IDO), an enzyme with immunosuppressive

properties is considered as a factor that impairs the antitumour immune response in mel-

anoma. In this study, we investigated the expression of IDO in sentinel nodes of melanoma

patients to determine its prognostic relevance.

Patients and methods: One hundred and sixteen melanoma patients were enrolled in this

study with a median follow-up time after diagnosis of 71 months. The expression of IDO

and forkhead box P3 (Foxp3) in the sentinel lymph nodes was determined by immunohis-

tochemistry and correlated with progression-free survival and overall survival. In 42

patients, regulatory T cells were investigated by flow cytometry.

Results: Cox regression survival analysis showed a significant negative effect of IDO expres-

sion on progression-free survival (p = 0.015) and overall survival (p = 0.010). High IDO

expression was correlated with a significant higher frequency of Foxp3-positive cells in

uninvaded lymph nodes (p = 0.016). The presence of IDO expression in the sentinel nodes

was not associated with an increased frequency of circulating regulatory T cells (Tregs)

but was significantly correlated with an increased mean fluorescence intensity of Cytotoxic

T-Lymphocyte Antigen 4 (CTLA-4) in Tregs (p = 0.019). After CD3CD28 stimulation, periphe-

ral blood mononuclear cells of patients with high IDO expression showed a lower produc-

tion of interferon-gamma (IFN-c) (p = 0.025).

Conclusions: This study points to an independent predictive role of IDO on survival, espe-

cially in melanoma patients with uninvolved sentinel nodes. Investigating IDO expression

in the sentinel nodes of melanoma patients may be a useful marker to pre-identify patients

with a less favourable prognosis in stage I and II disease.

� 2011 Elsevier Ltd. All rights reserved.

er Ltd. All rights reserved.

t of Dermatology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Tel.:

nt.be (R. Speeckaert).

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1. Introduction 2. Patients and methods

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppres-

sive enzyme physiologically expressed at the foetal–mater-

nal barrier, being involved in the tolerance of the foetus

by the maternal immune system. Increased IDO expression

has been observed in specific pathological conditions such

as viral infections and cancer.1 IDO is a rate-limiting

enzyme of the kynurenine pathway and catabolises trypto-

phan, an essential amino acid for the cell.2 Depletion of

tryptophan by the IDO activity in macrophages and den-

dritic cells (DCs) inhibits activation and proliferation of T

cells and natural killer (NK)) cells in the G1 phase of the

cell cycle. Moreover, tryptophan-depleted cells are more

vulnerable to apoptosis.3 Tryptophan catabolites (such as

L-kynurenine) also suppress the proliferation of activated T

cells.4–6

In melanoma, IDO expression has been reported in tumo-

ural cells and tumour-draining lymph nodes (TDLNs).7–9 Lee

et al.8 described IDO expression in antigen presenting cells

(APCs), located in the perisinusoidal and intrafollicular re-

gions of the lymph nodes of melanoma patients. These cells

were identified as mainly BDCA2+ plasmacytoid dendritic

cells (pDCs). Circulating IDO-expressing pDCs could not be

detected, suggesting that IDO expression in melanoma is a

local process limited to the primary tumour and TDLNs.10

Increasing evidence points to a prognostic role of IDO in

melanoma. Decreased serum tryptophan levels have been

associated with a worse prognosis in melanoma patients

and the expression of IDO by metastatic melanoma cells

has also been linked with impaired survival.9,11 IDO expres-

sion by immune cells in TDLNs has been suggested to predict

a worse outcome in melanoma.7,8 However, up till now, a

study to confirm the independent prognostic effect of

IDO expression by immune cells in the sentinel nodes of mel-

anoma patients was lacking and is of major importance

regarding the new insights in the key immunomodulating

role of IDO which seems an attractive target for

immunotherapy.12

IDO expression leads to a shift in lymphocytes to a regula-

tory T cell (Treg) phenotype.13 Regulatory T cells (Tregs) are an

extensively investigated subset of lymphocytes exerting

immunosuppressive activities. Several studies have shown a

higher prevalence of forkhead box P3 (Foxp3)+ Tregs in differ-

ent cancer types and the eradication of Tregs seems to be

associated with increased antitumoural responses.14 In mela-

noma, Tregs are increased around the primary lesion and in

the sentinel nodes.15,16 A recent study could not demonstrate

an independent prognostic effect of the frequency of Tregs in

primary melanoma lesions.16

In this study, we investigated the expression of IDO in the

sentinel nodes of melanoma patients to determine the prog-

nostic relevance and to correlate IDO expression with sys-

temic immunosuppressive factors. As IDO activity leads to a

shift in lymphocytes to a Treg phenotype, Foxp3 expression

in the lymph nodes and circulating Tregs were also

investigated.

2.1. Patients selection

One hundred and sixteen melanoma patients (46 males, 70 fe-

males) with a median age of 52 years at the time of diagnosis

(Table 1) were enrolled in this study. The study was approved

by the medical ethical committee of Ghent University Hospi-

tal. All patients signed written informed consent. Median fol-

low up after diagnosis was 71 months (Interquartile Range

(ICQ): 45–97). In 42 melanoma patients venous blood samples

were drawn in sterile ethylene diamine tetra-acetic acid

(EDTA) vacutainers (9 mL) within 4 years after the sentinel

procedure.

2.2. Immunohistochemistry

The immunohistochemical expression of IDO and Foxp3 was

investigated on paraffin-embedded sentinel nodes. The

immunohistochemical stainings were performed with the

standard avidin–biotin-peroxidase (ABC) protocol using

3-amino-9-ethylcarbazole (AEC) as chromogene on 4 lm thick

sections. Serial sections were incubated with a monoclonal

anti-Foxp3 (1/100, eBioscience, San Diego, USA) and a mono-

clonal anti-IDO antibody (clone 10.1, 1/200, Millipore, Billerica,

USA) for 1 h. For the staining with CD3 (RTU, Dako), CD4 (RTU,

Dako), CD31 (RTU, Dako) and CD34 (RTU, Dako) an incubation

time of 30 min was used.

The amount of IDO expression in non-metastatic areas

was automatically analysed on 3 hotspots (400·) with ImageJ

software (NIH, Bethesda, USA) and divided into two groups

according to their expression profile (almost none/weak ver-

sus strong IDO expression). The frequency of Foxp3 positive

cells was automatically counted with ImageJ software. Three

hotspots (400· magnification) were identified and the per-

centage of Foxp3 positive cells over the total counted nuclei

per field was determined.

2.3. Flow cytometry

Isolation of peripheral blood mononuclear cells (PBMCs) from

heparinized venous blood was performed by centrifugation

on a Ficoll-Hypaque gradient (GE Healthcare, Uppsala, Swe-

den). The cells were cryopreserved at )80 �C in heat-inacti-

vated foetal bovine serum (FBS) supplemented with 10%

dimethyl sulphoxide (DMSO) until analysis. Flow cytometry

was performed on PBMCs using membranous staining for

CD3 (PerCP, BD Bioscience, Erembodegem, Belgium), CD4

(FITC, BD Bioscience) and CD25 (APC-Cy7, Biolegend, San Die-

go, CA, USA) during 30 min on ice. After permeabilization and

fixation steps, the cells were stained for Foxp3 (APC, eBio-

science, San Diego, CA, USA) and Cytotoxic T-Lymphocyte

Antigen 4 (CTLA-4) (PE, BD Bioscience) during 20 min. Live/

dead staining was performed using an Aqua Dead Cell Stain

kit (Invitrogen, Merelbeke, Belgium). For cytokine analysis of

T helper (Th) cells, PBMCs were stimulated with 50 ng/mL

phorbol 12-myristate 13-acetate (PMA) (Fluka Biochemika,

Table 1 – Clinical characteristics of the study population.

Characteristic

Number of patients 116Age at time of diagnosis (years) 52 (IQR: 37–63)Males (%)/females 40% (n = 46)Location of primary melanoma

Head, neck (%) 6% (n = 7)Trunk (%) 34% (n = 40)Extremities (%) 60% (n = 69)

Evolution during follow upDisease progression (%) 28% (n = 32)Mortality (%) 18% (n = 21)

BreslowpT1 61.00 mm 10% (n = 12)pT2 1.01–2.00 mm 50% (n = 58)pT3 2.01–4.00 mm 31% (n = 36)pT4 >4.00 mm 9% (n = 10)

Ulceration (%) 25% (n = 29)Invaded sentinel nodes (%) 22% (n = 26)

2006 E U R O P E A N J O U R N A L O F C A N C E R 4 8 ( 2 0 1 2 ) 2 0 0 4 – 2 0 1 1

Buchs, Switzerland) and 750 ng/mL ionomycin (Invitrogen,

Merelbeke, Belgium) for 6 h. Brefeldin A (10 lg/mL) (Calbio-

chem, La Jolla, CA, USA) was added during the last 5 h of incu-

bation. Membrane staining with anti-CD3 (PerCP, BD

Biosciences, Erembodegem, Belgium) and anti-CD4 (APC-

Cy7, Biolegend, San Diego, CA, USA) antibodies was per-

formed. After permeabilization and fixation intracellular

cytokine production was detected by tumor necrosis factor-

alpha (TNF-a) (FITC, BD Biosciences, Erembodegem, Belgium),

interferon-gamma (IFN-c) (FITC, BD Biosciences, Erembodegem,

Belgium), IL-4 (PE, BD Biosciences, Erembodegem, Belgium),

IL-17a (APC, eBioscience, Vienna, Austria) antibodies. Dead

cells were excluded from analysis using a Live/Dead Fixable

Aqua Dead Cell Stain kit (Invitrogen, Merelbeke, Belgium).

For experiments depleting Tregs, PBMCs were incubated

with anti-CD25 Microbeads (Miltenyi Biotec, Leiden, The

Netherlands) for 15 min at 4 �C and purified on a MACS

column (Miltenyi Biotec, Leiden, The Netherlands). To analyse

cell proliferation, carboxyfluorescein succinimidyl ester

(CFSE) (500 ng/mL, Invitrogen, Merelbeke, Belgium) was added

and stimulation was performed with anti-CD2/CD3/CD28

beads (Treg inspector, Miltenyi Biotec, Leiden, The Netherlands)

for 5 days. Data acquisition of a minimum of 200,000 events

was performed on a LSR II (BD Biosciences, Erembodegem,

Belgium) and analysed with Flowjo 7.6.2 software (Treestar

Inc, San Carlos, CA, USA).

2.4. Physiologic stimulation of PBMCs

PBMCs were incubated overnight with CD3–CD28 beads (Invit-

rogen, Merelbeke, Belgium) at 37 �C in 10% CO2 and were

seeded in a medium containing Iscove’s Modified Dulbecco’s

Medium (IMDM) supplemented with 20% FBS and 10% gluta-

mine. Supernatant was harvested and analysed immediately

with multiplex bead array. Cytokine levels of IL-2, IL-10, TNF-a

and IFN-c (R&D Systems, Abingdon, UK) were measured using

a commercial kit (Human Fluorokine MAP Base Kit, R&D Sys-

tems). All measurements were performed according to the

manufacturer’s instructions. Cytokine analysis was per-

formed on a Bioplex 200 System (Bio-Rad, Hercules, CA, USA).

2.5. Cytospin

Cytospin slides were prepared by centrifugation of 200 micro-

litres of a cell suspension of PBMCs (1 · 106 cells/mL) during

5 min at 800g. Fixation was performed in acetone during

10 min at )20 �C. As a positive control, PBMCs were primed

with recombinant human IFN-c (50 ng/ml) (R&D Systems,

Abingdon, UK) and harvested at 72 h. The staining procedure

was similar to paraffin-embedded tissue.

2.6. Statistical analyses

The immunohistochemical expression patterns and flow

cytometry data were compared with Fisher’s Exact test, Spear-

man’s rank correlation test and the Mann–Whitney U-test.

Kaplan–Meier analysis and Cox regression analysis were car-

ried out to investigate the independent prognostic effect of

IDO and Foxp3 expression on melanoma-related mortality

and progression-free survival. All statistical analyses were

performed with SPSS 17.0 (SPSS Inc, Chicago, IL, USA). All val-

ues are expressed as median [interquartile range (IQR)]. Sta-

tistical significance was set at p < 0.05.

3. Results

3.1. IDO and Foxp3 expression in the sentinel node

IDO expression was observed in the cytoplasm of immune

cells mainly situated in the perisinusoidal areas of the lymph

nodes (Fig. 1A and C). More centrally in the lymph node,

CD31+ cells of high endothelial venules showed IDO expres-

sion (Fig. 1B and D). The expression pattern of IDO showed

an almost dichotomous distribution between lymph nodes

with strong expression (30.2%) and lymph nodes with almost

none or very little expression (69.8%). The ratio of strong ver-

sus none/weak IDO expression was significantly lower in in-

vaded sentinel nodes compared to negative lymph nodes

(p = 0.019). In patients with uninvolved sentinel nodes, Kap-

lan–Meier analysis showed a significant negative association

of strong IDO expression on progression-free survival

(p = 0.011) and overall survival (p = 0.021) (Fig. 2). Cox regres-

sion analysis (with adjustment for age, gender, Breslow thick-

ness, ulceration, anatomical location of the primary tumour

and sentinel involvement) showed a significant effect of IDO

expression on progression-free survival (p = 0.015) and overall

survival (p = 0.010). In patients with negative sentinel lymph

nodes, strong IDO expression was also independently associ-

ated with progression-free (p = 0.040) and overall survival

(p = 0.009) (Table 2).

Foxp3 staining showed a nuclear expression mainly in

lymphocytes of the paracortical zone of the lymph nodes

(Fig. 1E and F). Foxp3+ cells were located in the CD3+CD4+

areas of the sentinel nodes. The distribution patterns of

Foxp3+ cells were similar between invaded and non-invaded

lymph nodes. The percentage of Foxp3+ cells on the total T

cell population was significantly higher in invaded sentinel

nodes (median: 12%; IQR: 8.5–13.5%) compared to non-in-

vaded lymph nodes (median: 7%; IQR: 5–9%) (p < 0.001)

(Fig. 3A; Table 3). A significant positive association between

the frequency of Foxp3+ cells and IDO expression (p = 0.016)

Fig. 1 – Immunohistochemical pictures: weak expression of Indoleamine 2,3-dioxygenase (IDO) (A, B) and strong expression

of IDO (C, D) in the sentinel nodes; low frequency of forkhead box P3 (Foxp3) cells in a non-invaded sentinel node (E), high

frequency of Foxp3 cells in an invaded sentinel node (F).

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was found in patients with uninvolved sentinel nodes

(Fig. 3B). However, this association was not observed in in-

vaded lymph nodes. After adjustment for lymph node inva-

sion a significant association between Foxp3 expression and

Breslow thickness (p = 0.032) was detected. Kaplan–Meier

analysis showed a significant impaired survival (p = 0.007) in

patients with a high frequency of Foxp3+ cells (>10% of T cells)

compared to patients with a low frequency of Foxp3+ cells

(<10% of T cells). However, a multivariate Cox regression mod-

el showed no prognostic effect of Foxp3 expression on sur-

vival and progression-free survival.

3.2. Association of IDO expression with systemicimmunosuppressive markers

Because of the strong prognostic effect of IDO expression

on progression-free survival and overall survival, we

hypothesised that local IDO expression could be associated

with a systemic immunosuppressive climate. No IDO

expression was found in cytospins of PBMCs. In 42 patients,

flow cytometry was performed on PBMCs. Tregs were

marked as CD3+CD4+CD25+Foxp3+ cells and were found

with a median percentage of 4.99% (ICQ: 3.42–7.31). The

amount of Tregs was not different between patients with

low or high IDO expression. To detect a possible difference

in CTLA-4 expression between Tregs of patients with low

versus high sentinel IDO expression, the mean fluorescence

intensity (MFI) of CTLA-4 in Tregs was compared. In pa-

tients with uninvolved sentinel nodes (n = 35), CTLA-4-

expression on Tregs was significantly higher in patients

with a strong expression of IDO in the sentinel node

(n = 12) compared to the combined group of patients with

none to moderate IDO positivity (n = 23) (p = 0.019)

(Fig. 3C). Elevated CTLA-4 levels in Tregs were significantly

associated with higher Foxp3 levels (p = 0.004). The influ-

ence of high CTLA-4 expressing Tregs on TCR-induced pro-

liferation was investigated by comparing 4 samples with

high CTLA-4 in Tregs with 5 samples with low CTLA-4.

The percentage of proliferating PBMCs was decreased in pa-

tients with IDO expression in the sentinel node (mean:

44.96%) compared to the IDO negative group (mean:

56.94%). CD25-depletion could partly reverse this difference

between IDO+ patients (mean: 60.78%) and IDO) patients

(mean: 62.20%).

Fig. 2 – Kaplan–Meier analysis of progression-free-survival (left) and overall survival (right) according to IDO expression in

melanoma patients with negative sentinel nodes.

Table 2 – Cox regression survival analysis of Indoleamine 2,3-dioxygenase (IDO) expression in the sentinel nodes ofmelanoma patients.

IDO expression (none/little expression versus strong expression) Hazard ratio (95% CI) Wald v2 p-Value

Overall patient group (n = 116)a

End-point: survival 4.673 (1.46–14.93) 6.724 0.01End-point: progression-free survival 2.82 (1.23–6.45) 5.971 0.015

Patients with negative sentinel nodes (n = 90)a

End-point: survival 10.10 (1.76–58.82) 6.729 0.009End-point: progression-free survival 2.93 (1.05–8.197) 4.225 0.04

a Adjustment for age, gender, Breslow thickness, ulceration, anatomical location of the primary tumour and sentinel invasion.

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Cytokine secretion by PBMCs was analysed in the superna-

tant after stimulation with CD3CD28 beads for 14 h. The pro-

inflammatory cytokines IL-2, TNF-a, IFN-c and the anti-

inflammatory cytokine IL-10 were measured by multiplex

bead array. In patients with uninvolved sentinel nodes, IFN-

c levels (median: 319.37 pg/mL; ICQ range: 193.75–775.03) were

significantly lower in the group with strong IDO expression

versus none to low IDO expression (p = 0.025; median:

282.07 pg/mL versus 628.50 pg/mL) (Fig. 3D). IL-2, IL-10 and

TNF-a values were not different between the 2 subgroups

with different sentinel IDO expression.

Th1, Th2 and Th17 subsets were investigated by analysing

the number of TNF-a, IFN-c, IL-4 and IL17a producing

CD3+CD4+ cells. No difference was found in Th1 and Th17-

cytokine producing CD4+ cells, whereas a tendency to a high-

er percentage of IL-4 producing CD4+ cells was observed in the

IDO+ group [median % in IDO+ patients: 1.31% (0.97–2.49) ver-

sus IDO) patients: 0.87% (0.18–1.36)] although significance

was not reached (p = 0.058).

4. Discussion

Despite all current insights in prognostic factors for mela-

noma, this tumour can behave aggressively in all stages of

disease. There is a need to pre-identify the subgroup of

patients with a bad prognosis in order to develop adequate

management strategies. Immune escape could be one of the

mechanisms opening a way for an aggressive behaviour of

the tumour. Given the quest for effective immunotherapeutic

strategies, the in vivo implications of specific immune-sup-

pressing markers are interesting research topics.17 Increasing

evidence suggests that the production of IDO is a major

mechanism of immune escape in melanoma.13 We investi-

gated the impact of IDO and Foxp3 expression in the sentinel

nodes of melanoma patients on outcome and on the local and

systemic immunosuppressive climate.

In this study, enhanced IDO expression in the sentinel

node emerges as an independent prognostic factor as shown

by Cox regression survival analysis both with respect to over-

all survival as well as progression-free survival. IDO expres-

sion in the sentinel nodes may be a useful marker to pre-

identify a subset of patients in whom melanoma will behave

more aggressively. Hence, this subset of patients may benefit

from targeted immunomodulation (such as IDO inhibitors).

As expected, we found in analogy with Viguier et al.15, a

higher expression of Foxp3 in the sentinel nodes of mela-

noma patients with invaded sentinel nodes. Nonetheless, no

independent prognostic effect on overall survival or

progression-free-survival of Foxp3 expression could be

demonstrated.

Fig. 3 – Box-and-whisker plots showing the expression of Foxp3 according to sentinel lymph node involvement (A) and

comparing the expression of IDO in the sentinel node with Foxp3 expression in uninvolved sentinel nodes (B), with the mean

fluorescence intensity (MFI) of Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) in circulating regulatory T cells (Tregs) (C) and with

IFN-c values after CD3C28 stimulation of peripheral blood mononuclear cells (PBMCs) (D).

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In patients with IDO+ lymph nodes we detected in unin-

volved lymph nodes a higher frequency of Foxp3-expressing

cells and an enhanced MFI of CTLA-4 in circulating Tregs. This

suggests that IDO may be involved in creating a loco-regional

and perhaps also systemic immunosuppressive environment

early in the disease process which is supported by the worse

outcome in these patients. IDO production promotes the dif-

ferentiation of naıve T cells to Tregs and inhibits the conver-

sion of Foxp3+ cells to Th17 cells in tumour draining lymph

nodes.18–20 In addition, binding of B7-family receptors on

IDO+ pDCs with CTLA-4 on Tregs induces proliferation of

Tregs and leads to antigen-specific anergy.21,22 Furthermore,

the induction of IDO by CLTA-4 receptors on Tregs has been

demonstrated by in vitro and mice experiments.18 In mice,

specific deletion of CTLA-4 in Tregs resulted in a significantly

decreased IDO-expression by DCs in mesenteric lymph

nodes.23 The results of our study indicate that this mecha-

nism may also be involved in the induction of IDO in mela-

noma patients. Recently, CTLA-4 blockade with the

monoclonal antibody ipilimumab has been shown to induce

an overall survival benefit in melanoma patients.24 As com-

monly seen with targeted biological therapy, there is hetero-

geneity among CTLA-4 responders. Hence, IDO expression

levels in non-invaded sentinel lymph nodes might help to

select patients with the most potential benefit from anti-

CTLA-4 treatment.25

Decreased IFN-c secretion was found in PBMCs of pa-

tients with high IDO-expressing lymph nodes after

CD3CD28 stimulation. This finding may reflect a generalised

decreased immune reactivity against (tumoural) antigens

even in these early-stage patients. Sørensen et al.22 found

the presence of a substantial amount of IDO-specific T cells

in cancer patients and in healthy individuals which have

the capacity to eliminate tumour cells and immune cells

that produce IDO. High IFN-c levels can induce IDO-specific

T cells and in an IFN-c rich environment, IDO-specific T

cells show an enhanced immune reactivity.22 A decreased

induction of IFN-c may inhibit the generation and impair

the activity of IDO-specific T cells. This can result in a defi-

cient elimination of tumour cells and tolerance of immuno-

suppressive IDO-producing immune cells in the lymph

nodes. Future research focusing on genetic differences in

the IFN-c gene according to IDO expression could be inter-

esting as an association between a single nucleotide poly-

morphism [+874(T/A)] in the IFNc gene and the rate of

tryptophan metabolism was previously described.26 Several

polymorphisms are linked to a difference in induction

capacity of IFN-c production by lymphocytes.27

Table 3 – IDO and forkhead box P3 (Foxp3) expression according to the clinicopathological characteristics.

Patient group Characteristic Number ofpatients

Number ofdeaths at

end of study

IDO low IDOhigh

Percentage (%)with high IDOexpression (%)

Foxp3 low(<10% of T cells)

Foxp3 high(>10% of T cells)

Percentage (%)with high Foxp3

expression (>10% of T cells) (%)

All patients Males 46 12 30 16 34.8 32 14 30.4Females 70 9 51 19 27.1 49 21 30.0H&N 7 3 4 3 42.9 6 1 14.3Trunk 40 8 26 14 35.0 19 21 52.5Limbs 69 10 51 18 26.1 55 14 20.3BT <1 12 0 7 5 41.7 10 2 16.7BT 1–1.99 58 7 43 15 25.9 42 16 27.6BT2.1.99–3.99 36 9 23 13 36.1 23 13 36.1BT4+ 10 5 8 2 20.0 5 5 50.0Ulceration no 87 12 62 25 28.7 64 23 26.4Ulceration yes 29 9 19 10 34.5 17 12 41.4

Patients withnegative sentinelnodes

Males 34 6 19 15 44.1 28 6 17.6Females 56 4 39 17 30.4 44 12 21.4H&N 6 2 3 3 50.0 5 1 16.7Trunk 27 3 15 12 44.4 16 11 40.7Limbs 57 5 40 17 29.8 51 6 10.5BT <1 12 0 7 5 41.7 10 2 16.7BT 1–1.99 47 5 33 14 29.8 39 8 17.0BT2 1.99–3.99 27 5 15 12 44.4 19 8 29.6BT4+ 4 0 3 1 25.0 4 0 0.0Ulceration no 74 8 50 24 32.4 59 15 20.2Ulceration yes 16 2 8 8 50.0 14 2 12.5

Patients withpositive sentinelnodes

Males 12 6 11 1 8.3 4 8 66.6Females 14 5 12 2 14.3 5 9 64.3H&N 1 1 1 0 0.0 1 0 0.0Trunk 13 5 11 2 15.4 3 10 76.9Limbs 12 5 11 1 8.3 4 8 66.7BT <1 0 0 0 0 0 0BT 1–1.99 11 2 10 1 9.1 3 8 72.7BT2.1.99–3.99 9 4 8 1 11.1 4 5 55.5BT4+ 6 5 5 1 16.7 1 5 83.3Ulceration no 13 4 12 1 7.7 5 8 61.5Ulceration yes 13 7 11 2 15.4 3 10 76.9

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E U R O P E A N J O U R N A L O F C A N C E R 4 8 ( 2 0 1 2 ) 2 0 0 4 – 2 0 1 1 2011

In conclusion, this study highlights the role of IDO as an

independent prognostic factor, which corroborates its known

immunosuppressive function. Immunohistochemical IDO

expression in the lymph nodes might be used as a prognostic

factor to identify the subgroup of melanoma patients with

negative sentinel nodes that nonetheless are at high risk for

the development of metastatic disease. Pharmacological

blockade of IDO together with CTLA-4 inhibition could be of

particular relevance in patients with IDO positive sentinel

nodes.28 Future research is needed to clarify further the com-

plex immune network associated with IDO expression and

determine whether melanoma patients with IDO expression

in the sentinel node show different response rates to anti-

gen-specific cancer immunotherapy.

Conflict of interest statement

None declared.

Acknowledgments

Financial support: R. Speeckaert is funded by a BOF Grant

from the Ghent University, Belgium. M. Van Gele is a postdoc-

toral research fellow of the Fund for Scientific Research-Flan-

ders (Belgium).

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