Immune response in melanoma: an in-depth analysis of the primary tumor and corresponding sentinel lymph node.

Abstract

The sentinel lymph node is the initial site of metastasis. Downregulation of antitumor immunity has a role in nodal progression. Our objective was to investigate the relationship between immune modulation and sentinel lymph node positivity, correlating it with outcome in melanoma patients. Lymph node/primary tissues from melanoma patients prospectively accrued and followed at New York University Medical Center were evaluated for the presence of regulatory T cells (Foxp3(+)) and dendritic cells (conventional: CD11c(+), mature: CD86(+)) using immunohistochemistry. Primary melanoma immune cell profiles from sentinel lymph node-positive/-negative patients were compared. Logistic regression models inclusive of standard-of-care/immunological primary tumor characteristics were constructed to predict the risk of sentinel lymph node positivity. Immunological responses in the positive sentinel lymph node were also compared with those in the negative non-sentinel node from the same nodal basin and matched negative sentinel lymph node. Decreased immune response was defined as increased regulatory T cells or decreased dendritic cells. Associations between the expression of these immune modulators, clinicopathological variables, and clinical outcome were evaluated using univariate/multivariate analyses. Primary tumor conventional dendritic cells and regression were protective against sentinel lymph node metastasis (odds ratio=0.714, 0.067; P=0.0099, 0.0816, respectively). Antitumor immunity was downregulated in the positive sentinel lymph node with an increase in regulatory T cells compared with the negative non-sentinel node from the same nodal basin (P=0.0005) and matched negative sentinel lymph node (P=0.0002). The positive sentinel lymph node also had decreased numbers of conventional dendritic cells compared with the negative sentinel lymph node (P<0.0001). Adding sentinel lymph node regulatory T cell expression improved the discriminative power of a recurrence risk assessment model using clinical stage. Primary tumor regression was associated with prolonged disease-free (P=0.025) and melanoma-specific (P=0.014) survival. Our results support an assessment of local immune profiles in both the primary tumor and sentinel lymph node to help guide therapeutic decisions.

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Immune response in melanoma: an in-depth
analysis of the primary tumor and
corresponding sentinel lymph node
Michelle W Ma1,2,*, Ratna C Medicherla1,2,*, Meng Qian3, Eleazar Vega-Saenz de Miera1,2,
Erica B Friedman2,4, Russell S Berman2,4,5, Richard L Shapiro2,4,5, Anna C Pavlick1,2,5,6,
Patrick A Ott2,5,6, Nina Bhardwaj1,2,5,6,7, Yongzhao Shao2,3, Iman Osman1,2,5and
Farbod Darvishian2,7
1Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York,
NY, USA;2Interdisciplinary Melanoma Cooperative Group, New York University School of Medicine,
New York, NY, USA;3Division of Biostatistics, New York University School of Medicine, New York, NY, USA;
4Department of Surgery, New York University School of Medicine, New York, NY, USA;5New York University
Cancer Institute, New York, NY, USA;6Department of Medicine, New York University School of Medicine,
New York, NY, USA and7Department of Pathology, New York University School of Medicine, New York,
NY, USA
The sentinel lymph node is the initial site of metastasis. Downregulation of antitumor immunity has a role in
nodal progression. Our objective was to investigate the relationship between immune modulation and sentinel
lymph node positivity, correlating it with outcome in melanoma patients. Lymph node/primary tissues from
melanoma patients prospectively accrued and followed at New York University Medical Center were evaluated
for the presence of regulatory T cells (Foxp3þ) and dendritic cells (conventional: CD11cþ, mature: CD86þ)
using immunohistochemistry. Primary melanoma immune cell profiles from sentinel lymph node-positive/-
negative patients were compared. Logistic regression models inclusive of standard-of-care/immunological
primary tumor characteristics were constructed to predict the risk of sentinel lymph node positivity.
Immunological responses in the positive sentinel lymph node were also compared with those in the negative
non-sentinel node from the same nodal basin and matched negative sentinel lymph node. Decreased immune
response was defined as increased regulatory T cells or decreased dendritic cells. Associations between the
expression of these immune modulators, clinicopathological variables, and clinical outcome were evaluated
using univariate/multivariate analyses. Primary tumor conventional dendritic cells and regression were
protective against sentinel lymph node metastasis (odds ratio¼0.714, 0.067; P¼0.0099, 0.0816, respectively).
Antitumor immunity was downregulated in the positive sentinel lymph node with an increase in regulatory
T cells compared with the negative non-sentinel node from the same nodal basin (P¼0.0005) and matched
negative sentinel lymph node (P¼0.0002). The positive sentinel lymph node also had decreased numbers of
conventional dendritic cells compared with the negative sentinel lymph node (Po0.0001). Adding sentinel
lymph node regulatory T cell expression improved the discriminative power of a recurrence risk assessment
model using clinical stage. Primary tumor regression was associated with prolonged disease-free (P¼0.025)
and melanoma-specific (P¼0.014) survival. Our results support an assessment of local immune profiles in both
the primary tumor and sentinel lymph node to help guide therapeutic decisions.
Modern Pathology advance online publication, 16 March 2012; doi:10.1038/modpathol.2012.43
Keywords: dendritic cells; lymphatic metastases; melanoma; regulatory T cells; sentinel lymph node biopsy
Received 12 December 2011; revised 25 January 2012; accepted 25 January 2012; published online 16 March 2012
Correspondence: Dr F Darvishian, MD, Department of Pathology, New York University School of Medicine, 530 First Avenue, Skirball 7N,
New York, NY 10016, USA.
E-mail: Farbod.Darvishian@nyumc.org
*These authors contributed equally to this work.
This work was presented in part at the Annual Meeting of the American Society of Clinical Oncology, 3–7 June 2011, Chicago, IL.
Modern Pathology (2012), 1–11
& 2012 USCAP, Inc. All rights reserved 0893-3952/12 $32.00
1
www.modernpathology.org

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Sentinel lymph node status continues to be the most
important prognostic factor in melanoma not only
for recurrence1but also overall survival.2,3Melanoma
patients with thick (41.00mm) and ulcerated
primary tumors, in particular, are at high risk for
occult nodal metastasis and warrant a sentinel
lymph node biopsy to evaluate for the presence of
nodal disease.2Sentinel lymph node metastasis,
however,doesoccur
(r1.00mm) melanomas4such that additional selec-
tion criteria may improve the sentinel lymph node
positivity risk-stratification model based on thick-
ness and ulceration alone.
Immune cell populations in the primary tumor
reflect the host immune response, and there is
evidence to suggest that the immunophenotype of
this immune response is predictive of sentinel
lymph node status not only in melanoma3,5–10but
also in colorectal,11esophageal,12gastric,13and
papillary thyroid cancer.14Primary tumor-infiltrat-
ing lymphocytes have an important role in the
antitumor T cell response that mediates regression,
and the absence of tumor-infiltrating lymphocytes
and regression have both been shown to predict
sentinel lymph node positivity in melanoma.3,5–10
Theprognostic relevance of different primary
tumor T cell subsets, however, has not yet been
examined in melanoma even though studies in other
cancers have demonstrated the clinical value of
characterizing tumor-infiltrating lymphocyte sub-
populations,suchas
Foxp3þregulatory Tcell subset, in predicting nodal
metastasis.11–14
T cell
require the antigen-presenting capacity of dendritic
cells, including the immunogenic CD11cþconven-
tional dendritic cells and the tolerogenic CD123þ
plasmacytoid dendritic cells. Dendritic cell subsets
may therefore also predict sentinel lymph node
positivity in melanoma, and one study in melanoma
has already shown an association between primary
tumor CD123þplasmacytoid dendritic cells and
clinical outcome.15
Primary melanoma immune markers warrant
further investigation as potential predictors of
sentinel lymph node status, and given the impact
of primary tumor-derived cytokines on the immuno-
logical status of the sentinel lymph node16and the
importance of the pathological status of the sentinel
lymph node,1–3additional prognostic information
maybegainedby an
terization of the sentinel lymph node as well.
In this study, our objective was to first examine the
relationship between the immune profile of the
primary melanoma and sentinel lymph node posi-
tivity and then to compare the immunophenotype of
the immune response in positive sentinel lymph
nodes with that in negative sentinel lymph nodes
and negative non-sentinel nodes from the same
nodal basin and to correlate it with clinical outcome
in a cohort of prospectively-accrued cutaneous
melanoma patients.
in patients withthin
theimmunosuppressive
responses, furthermore,
immunologicalcharac-
Patients and methods
Study Population
Lymph node and primary melanoma tissues were
retrieved from patients enrolled in the Interdiscipli-
nary Melanoma Cooperative Group (IRB#10362),17a
prospectively collected clinicopathological-biospeci-
men database at New York University Langone
Medical Center (August 2002 to September 2009).
Informed consent was obtained from patients at the
time of enrollment, and all demographic, clinico-
pathological, and follow-up data were recorded
prospectively.
Patients with available sentinel lymph node biopsy
specimens were identified, and sentinel lymph node-
positive patients were matched to sentinel lymph
node-negative patients for age at initial melanoma
diagnosis, gender, primary tumor thickness (mm),
and ulceration status. Other clinicopathological
features collected included primary tumor mitotic
rate (mitoses/mm2), histological subtype, anatomic
site, and American Joint Committee on Cancer stage
at pathological diagnosis. Negative non-sentinel node
tissues were obtained from all sentinel lymph node-
positive patients as well from the same nodal basin as
the positive sentinel lymph node.
Assessment of Foxp3, CD11c, and CD86 Expression
Immunohistochemistry was performed using mouse
anti-human Foxp3 clone 236A/E7 (eBioscience, San
Diego, CA, USA), CD11c clone 5D11 (Novocastra
Laboratories, Newcastle upon Tyne, UK) and CD86
(R&D Systems, Minneapolis, MN, USA) on formalin
fixed, paraffin embedded lymph node and primary
melanoma tissues to detect regulatory T cells
(Foxp3þ), conventional dendritic cells (CD11cþ),
and mature dendritic cells (CD86þ), respectively.
Anti-human CD123 clone BR4MS (Novocastra) was
also used to identify plasmacytoid dendritic cells in
a subset of sentinel lymph node tissues. In brief,
afterdeparaffinization
induced epitope retrieval was performed in 0.01M
citrate buffer (pH 6.0) for Foxp3, CD11c, and CD86
in a 1200-W microwave oven at 100% power for
20min. Sections were then cooled in tap water for
5min, quenched in hydrogen peroxide for 30min,
washed with PBS, and incubated with blocking
serum (VECTASTAIN Elite ABC Kit–Mouse IgG,
Vector Laboratories, Burlingame, CA, USA) for
30min followed by each primary antibody diluted
in buffer (Foxp3, 1:500; CD11c, 1:50; CD86, 1:400) at
room temperature for 1h and at 41C overnight.
Slideswerewashedin
with diluted biotinylated secondary antibodies
(horse anti-mouse at 1:500 for both Foxp3- and
CD11c-stained sections; horse anti-goat at 1:100 for
CD86-stained sections, Vector Laboratories) for 1h.
Avidin-biotinylated horseradish peroxidase com-
plexes diluted at 1:500 (ABC Reagent, Vector
and rehydration,heat-
bufferandincubated
Immune response in melanoma
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Laboratories) were added, and complexes were
visualized with diaminobenzidine (DAB substrate
kit, Vector Laboratories). Slides were then washed in
distilled water, counterstained with hematoxylin,
dehydrated, and mounted with permanent media.
Appropriate positive and negative controls were
included with the study sections.
An attending pathologist (FD) who was blinded
to patients’ clinical data scored Foxp3, CD11c,
CD86, and CD123 expression as the absolute number
of positively-stained immune cells demonstrating
characteristic T cell and dendritic cell morphology,
respectively, in a representative high-power field
(HPF; 0.2mm2) that was selected by scanning each
slide at ?40 to find the field with the highest
antibody expression. Tumor involvement in each
specimen was also assessed semi-quantitatively as
the percentage of all cells using the corresponding
hematoxylin and eosin stained-section.
Statistical Analysis
Descriptive statistics were used to summarize demo-
graphicand primarytumor
melanoma patients. Distributional comparison of
continuous variables between the sentinel lymph
node-positive and sentinel lymph node-negative
groups was made using the two-sided Wilcoxon–
Mann–Whitney test. Distributional comparison of
continuous variables between the positive sentinel
lymph node and negative non-sentinel node from
the same nodal basin was made using the paired
t-test for variables that approximately follow the
normal distribution. The w2test, Fisher’s exact test,
and Armitage trend test were used to compare
independent proportions for categorical variables.
Logistic regression models were used to assess the
significance of predictors and to calculate the odds
ratios with or without adjustments of other covari-
ates/factors. The area under the receiver operating
characteristic curve was calculated as an indication
of the discriminative power of the logistic predictive
models. Statistical significance of tests was claimed
when P-values were o5% (Po0.05). In survival
analysis, Kaplan–Meier curves and log-rank tests
were used to assess the differential survival profiles
of the low- and high-risk groups. Statistical analyses
were conducted using SAS and the statistical
software R.
characteristics of
Results
Sentinel lymph node tissue was available for ana-
lysis from 84 melanoma patients. In all, 31 sentinel
lymph node-positive patients were matched by
age at pathological diagnosis, gender, primary tumor
thickness, and ulceration status to 53 sentinel
lymph node-negative patients. Primary tumor thick-
ness and ulceration status were similar between the
two groups (median thickness: 2.50mm vs 2.20mm,
P¼0.750; ulceration present: 35% vs 36%, P¼0.923,
respectively; Table 1). There was no significant differ-
ence in age at initial melanoma diagnosis or gender
as well primary tumor mitotic rate, histological
subtype, and anatomic site between sentinel lymph
node-positive and -negative patients (P40.05; Table 1).
Twenty-three patients recurred during follow-up
(median: 3.6 years for the whole cohort, 3.8 years
for survivors): 13/31 (42%) sentinel lymph node-
positive patients and 10/53 (19%) sentinel lymph
node-negative patients.
In all, 54 (64%) of the 84 patients also had primary
melanoma specimens available for analysis: 23
sentinel lymph node-positive and
lymph node-negative patients. Median primary
tumor thickness was similar between sentinel
lymph node-positiveand
(2.25mm vs 2.30mm, P¼0.92; Table 1). There were
also no significant differences between these two
groups in age at pathological diagnosis, gender,
primarytumorulceration
histological subtype, or anatomic site (P40.05;
Table 1). Sentinel lymph node-positive patients
did, however, have primary tumors with a signifi-
cantly higher tumor volume (mean: 63% vs 46%,
P¼0.043) and a lower number of CD11cþconven-
tional dendritic cells/HPF (mean: 9 vs 16, P¼0.054)
compared with those from sentinel lymph node-
negative patients as well as a decreased proportion
of primaries with regression (9% vs 35%, P¼0.028)
(Table 2). Yet, no significant difference in the pre-
sence of tumor-infiltrating lymphocytes, the number
of regulatory T cells or mature dendritic cells/HPF,
or solar elastosis were observed between the two
groups (P40.05; Table 2).
31sentinel
-negative patients
status,mitotic rate,
The Immune Profile of Primary Melanomas Predicts
Sentinel Lymph Node Positivity
Three different logistic regression models for pre-
dicting sentinel lymph node positivity are shown in
Table 3. Model 1 includes primary tumor thickness
and ulceration status, but as neither covariate inde-
pendently predicts sentinel lymph node status, this
model has a limited discriminatory accuracy with
an area under the receiver operating characteristic
curve equal to 0.5520 (Figure 1a). Model 2 is based
solely on the immune profile of the primary
melanoma (tumor-infiltrating lymphocytes, Foxp3þ
regulatory T cells, CD11cþconventional dendritic
cells, CD86þmature dendritic cells, regression, and
solar elastosis), and its discriminatory accuracy is
better with an area under the curve equal to 0.8205
(Figure 1b). Both primary tumor conventional
dendritic cells and regression are protective against
lymph node metastasis, but only conventional
dendritic cells are significantly associated with
sentinel lymph node status (odds ratio¼0.853,
0.158; P¼0.0092, 0.0698, respectively). Model 3
thencombinesstandard-of-careprimarytumor
Immune response in melanoma
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characteristics and markers of the immune response
to achieve an even higher discriminatory accu-
racy with an area under the curve equal to 0.9158
(Figure 1c).
Primarytumorconventional
continueto beprotective
P¼0.0099), and a rich network of CD11cþcells
can be seen in the primary melanoma from a
sentinel lymph node-negative patient (Figures 1d–f)
in contrast to the few CD11cþcells present in the
primary tumor from a sentinel lymph node-positive
patient (Figures 1g–i). Regression remains protective
against lymph node progression (odds ratio¼0.067)
as wellbut only trends
(P¼0.0816) as in the previous model. Increasing
age at pathological diagnosis is also protective (odds
ratio¼0.942, P¼0.0548), whereas a higher tumor
volume, the presence of tumor-infiltrating lympho-
cytes, and an increased proportion of mature
dendritic
ratio¼0.714,
cells
(odds
towardssignificance
dendritic cells are all risk factors for sentinel lymph
node positivity (odds ratio¼1.031, 10.790, 1.350;
P¼0.0546, 0.0451, 0.0330, respectively). There is no
concordance, however, between the immune profile
of the primary melanoma and the sentinel lymph
node (Supplementary Table 1).
The Immunologic Balance in the Positive Sentinel
Lymph Node Is Shifted Towards Tolerance
Positive sentinel lymph nodes have a tolerogenic
immune profile compared with negative sentinel
lymph nodes with the latter containing signifi-
cantly fewer immunosuppressive Foxp3þregula-
tory T cells/HPF (median: 45 vs 80, P¼0.0002;
Figures 2a–c) and more immunogenic CD11cþcon-
ventional dendritic cells/HPF (median: 35 vs 20,
P¼0.00002; Figures 2d–f). Yet, the number of CD86þ
Table 1 Demographic and primary melanoma features of patients who underwent a sentinel lymph node biopsy
Sentinel lymph node tissue availablePrimary tumor tissue available
Sentinel lymph
node-positive
N¼31
Sentinel lymph
node-negative
N¼53
P-valueSentinel lymph
node-positive
N¼23
Sentinel lymph
node-negative
N¼31
P-value
Age at pathological diagnosis (years)
Median (range)
0.1450.21
54 (28–79) 65 (29–86) 62 (28–79)64 (34–84)
Gender
Male, n (%)
Female, n (%)
0.3580.44
19 (61)
12 (39)
27 (51)
26 (49)
15 (65)
8 (35)
17 (55)
14 (45)
Primary tumor thickness (mm)
Median (range)
0.750 0.92
2.50 (0.90–20)2.20 (0.80–12) 2.25 (0.90–20)2.30 (0.80–12)
Primary tumor ulceration status
Absent, n (%)
Present, n (%)
0.9230.38
20 (65)
11 (35)
34 (64)
19 (36)
16 (70)
7 (30)
18 (58)
13 (42)
Primary tumor mitotic rate (mitoses/mm2)
0, n (%)
Z1, n (%)
Unclassified, n (%)
0.14 0.38b
1 (3)
29 (94)
1 (3)
8 (15)
44 (83)
1 (2)
1 (4)
22 (96)
0 (0)
4 (13)
27 (87)
0 (0)
AJCC stage at pathological diagnosis
I, n (%)
II, n (%)
III, n (%)
o0.0001
o0.0001
0 (0)
0 (0)
31 (100)
21 (40)
31 (58)
1 (2)a
0 (0)
0 (0)
23 (100)
11 (35)
20 (65)
0 (0)
Primary tumor histological subtype
Superficial spreading melanoma, n (%)
Nodular melanoma, n (%)
Other melanoma, n (%)
Unclassified, n (%)
0.1461b
9 (29)
17 (55)
4 (13)
1 (3)
19 (36)
28 (53)
5 (9)
1 (2)
8 (35)
12 (52)
3 (13)
0 (0)
10 (32)
17 (55)
4 (13)
0 (0)
Primary tumor anatomic site
Head/neck, n (%)
Axial, n (%)
Extremity, n (%)
0.2800.062b
4 (13)
10 (32)
17 (55)
3 (6)
25 (47)
25 (47)
4 (17)
5 (22)
14 (61)
3 (10)
17 (55)
11 (35)
Abbreviation: AJCC, American Joint Committee on Cancer.
Characteristics for the entire cohort (N¼84) and the subset with available primary tissue for analysis (N¼54) are listed separately.
aSatellites without metastatic nodes.
bBy Fisher’s exact test.
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mature dendritic cells is significantly decreased in
the negative sentinel lymph node compared with
the positive sentinel lymph node (median: 20 vs 36,
P¼0.0005; Figures 2g–i). A subset analysis of those
sentinel lymph node-positive cases with the highest
number of CD86þmature dendritic cells, how-
ever, showed that the immunosuppressive CD123þ
plasmacytoid dendritic cell subset exceeded the
immunogenic CD11cþconventional dendritic cell
subset by up to five fold (Supplementary Figure 1).
The immunological balance in the positive sentinel
lymph node compared with that in the negative non-
sentinel node from the same nodal basin is similarly
shifted away from an antitumor immune response
with its increased number of Foxp3þregulatory
T cells (P¼0.0005; Figures 3a, b) and decreased
number of CD11cþ
conventional dendritic cells
(P¼0.059; Figures 3c, d). CD86þmature dendritic
cells are likewise increased in the positive sentinel
lymph node compared with the negative non-
sentinel node from the same nodal basin (P¼0.06;
Figures 3e, f) as in the previous comparison.
Table 3 Logistic regression models for predicting sentinel lymph node positivity in melanoma patients
ModelCovariatesOdds ratio95% Confidence intervalP-value
1Primary tumor thickness (mm)
Primary tumor ulceration (present vs absent)
1.076
0.801
0.927–1.248
0.288–2.228
0.3355
0.6709
2Primary tumor-infiltrating lymphocytes (present vs absent)
Primary tumor Foxp3+regulatory T cells (#/HPF)
Primary tumor CD11c+conventional dendritic cells (#/HPF)
Primary tumor CD86+mature dendritic cells (#/HPF)
Primary tumor regression (present vs absent)
Primary tumor solar elastosis (present vs absent)
2.787
1.004
0.853
1.133
0.158
0.852
0.530–14.648
0.980–1.030
0.756–0.961
0.970–1.324
0.022–1.161
0.215–3.372
0.2261
0.7276
0.0092
0.1143
0.0698
0.8190
3 Primary tumor thickness (mm)
Primary tumor ulceration (present vs absent)
Percentage of tumor cells in primary (%)
Primary tumor-infiltrating lymphocytes (present vs absent)
Primary tumor CD11c+conventional dendritic cells (#/HPF)
Primary tumor CD86+mature dendritic cells (#/HPF)
Primary tumor regression (present vs absent)
Age at pathological diagnosis (years)
1.245
0.151
1.031
10.790
0.714
1.350
0.067
0.942
0.834–1.860
0.016–1.457
0.999–1.064
1.053–110.552
0.553–0.923
1.024–1.779
0.003–1.402
0.886–1.001
0.2831
0.1021
0.0546
0.0451
0.0099
0.0330
0.0816
0.0548
Abbreviation: HPF, high-power field.
Bold values are significant P-values.
Table 2 Immune profile of the primary melanoma in sentinel lymph node-positive vs sentinel lymph node-negative patients
Sentinel lymph
node-positive
N¼23
Sentinel lymph
node-negative
N¼31
P-value
Percentage of tumor cells in primary (%)
Mean; median
0.043
63; 7046; 40
Primary tumor-infiltrating lymphocytes
Absent, n (%)
Present, n (%)
0.85
6 (26)
17 (74)
9 (29)
22 (71)
Primary tumor Foxp3+regulatory T cells (#/HPF)
Mean; median
0.87
41; 35 44; 35
Primary tumor CD11c+conventional dendritic cells (#/HPF)
Mean; median
0.054
9; 8 16; 12
Primary tumor CD86+mature dendritic cells (#/HPF)
Mean; median
0.12
5; 4 4; 3
Primary tumor regression
Absent, n (%)
Present, n (%)
0.028a
21 (91)
2 (9)
20 (65)
11 (35)
Primary tumor solar elastosis
Absent, n (%)
Present, n (%)
0.18
16 (70)
7 (30)
16 (52)
15 (48)
Abbreviation: HPF, high-power field.
aBy Fisher’s exact test.
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Sentinel Lymph Node and Primary Tumor Immune
Profiles Contribute to Recurrence Risk
Sentinel lymph node positivity alone is associated
with an increased risk of recurrence (odds ratio¼
3.106, P¼0.0250). The addition of sentinel lymph
node regulatory Tcells to a logistic regression model
with clinical stage alone as a predictor of recurrence
improves its discriminative power, increasing its
area under the receiver operating characteristic
curve from 0.6871 to 0.7598. Primary tumor immune
markers contribute to recurrence risk as well, and
the presence of regression, in particular, is asso-
ciated not only with prolonged progression-free
survival (P¼0.025; Figure 4a) but also longer
melanoma-specific survival (P¼0.014; Figure 4b).
Discussion
Our study supports the crucial role of the host
immune response in both melanoma progression
and clinical outcome. Data suggest that the immune
profile of the primary melanoma predicts sentinel
lymph node positivity and that the presence of
primary tumor regression is a favorable prognostic
factor in thick (42.0mm) melanoma patients.
Modulation of antitumor immunity occurs in the
Figure 1 Immune profile of primary melanomas predicts sentinel lymph node positivity. Receiver operating characteristic curves
for three risk stratification models to predict sentinel lymph node positivity in melanoma patients: (a) Model 1, (b) Model 2, and
(c) Model 3. (d) Hematoxylin and eosin-stained primary tissue section (?100) from a sentinel lymph node-negative patient whose
primary melanoma demonstrates strong immunoreactivity for CD11c (?100, e). (f) High-power view (?400) showing a dense network
of CD11cþcells exhibiting characteristic dendritic cell morphology. (g) Primary melanoma section stained with hematoxylin and
eosin (?100) from a patient with a positive sentinel lymph node whose primary tumor illustrates weak CD11c immunostaining
(?100, h; ?400, i).
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sentinel lymph node as well, and the degree of
immunosuppression in the sentinel lymph node
microenvironment as measured by the number of
Foxp3þregulatory T cells, CD11cþconventional
dendritic cells, and CD86þmature dendritic cells,
varies with the extent of tumor involvement.
Highly tolerogenic microenvironments are asso-
ciated with advanced disease, and the immuno-
phenotype of the immune response in the sentinel
lymph node provides additional, independent prog-
nostic information to determine recurrence risk.
Primary tumor immune markers have previously
been shown to predict sentinel lymph node meta-
stasis in melanoma,3,5–10and our data provide
further evidence in support of the predictive value
of primary tumor regression and tumor-infiltrating
lymphocytes as it relates to sentinel lymph node
positivity. Regression results from a T cell immune
response, and our study suggests that its presence is
associated with a decreased risk of nodal progres-
sion, which is consistent with data from other mela-
noma studies.3,5–7Yet, some studies contend that
primary tumor regression is a risk factor for sentinel
lymph node positivity, albeit in patients with thin
(r1.00mm) melanomas who are then selected to
undergo a sentinel lymph node biopsy.18,19It is
Figure 2 Immunological balance in the positive sentinel lymph node is shifted towards tolerance. Consecutive negative sentinel lymph
node and positive sentinel lymph node sections were each stained for Foxp3, CD11c, and CD86. Representative negative sentinel lymph
node (a) illustrates weak Foxp3 immunoreactivity compared with the positive sentinel lymph node (b; ?100), and corresponding box
plots (c) show a significant increase in the number of Foxp3þregulatory T cells/HPF in the positive sentinel lymph node. Prominent
CD11c immunostaining is shown in the consecutive negative sentinel lymph node section (d) as compared with that in the positive
sentinel lymph node (e; ?100), and box plots illustrate this significant decrease in the number of CD11cþconventional dendritic cells/
HPF in the positive sentinel lymph node (f). Less intense CD86 immunostaining in the next consecutive negative sentinel lymph node
section (g) is shown as well in comparison to the increased density of CD86þcells with dendritic cell morphology in the positive sentinel
lymph node (h; ?100), a finding also represented in the corresponding box plots (i).
Immune response in melanoma
MW Ma et al
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important to recognize that our study cohort had
thicker melanomas (median: 2.28mm), such that our
findings are not disputing the claim that regres-
sion in thin melanomas is a poor prognostic
factor.18,19There is, however, an apparent contra-
diction between our study and others regarding the
relationship betweenprimary
lymphocytes and sentinel lymph node metastasis.
Much of the evidence supports the protective role of
tumor-infiltrating lymphocytes8–10in contrast to our
finding that tumor-infiltrating lymphocytes increase
the risk of nodal progression. These previous studies
did not characterize tumor-infiltrating lymphocyte
subpopulations though, which include cytotoxic
T cells as well as immunosuppressive regulatory
T cells. Ours is the first to examine the predictive
value of primary tumor regulatory T cells for
sentinel lymph node positivity, and although our
final model of sentinel lymph node positivity risk
excludes regulatory T cells, their presence likely
accounts for the observed increased risk of tumor-
infiltrating lymphocytes. Regulatory T cell differen-
tiation is promoted by the immunosuppressive
cytokine interleukin-10 that also prevents the func-
tion of conventional dendritic cells, a dendritic cell
subset evaluated for the first time in this study as it
tumor-infiltrating
Figure 3 Foxp3þregulatory T cells decrease progressively in the positive nodal basin. Consecutive sections from a representative
positive sentinel lymph node and a negative non-sentinel node from the same nodal basin were stained for Foxp3, CD11c, and CD86.
Strong immunoreactivity for Foxp3 is shown in the positive sentinel lymph node (a) as compared with the negative non-sentinel node
(b; ?100). Lower levels of CD11c immunopositive cells with characteristic dendritic cell morphology are found in the positive sentinel
lymph node (c) in comparison to the negative non-sentinel node (d) (?100), whereas mildly elevated levels of CD86 expression are seen
in the positive sentinel lymph node (e) compared with the negative non-sentinel node (f; ?100).
Figure 4 Primary melanoma regression is associated with pro-
longed survival. Kaplan–Meier estimates of (a) progression-free
survival and (b) melanoma-specific survival stratified according
to primary tumor regression.
Immune response in melanoma
8
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Page 9

relates to sentinel lymph node metastasis. The
presence of CD11cþconventional dendritic cells
protects againstnodal
consistent with findings from a recent study by our
group demonstrating that primary melanoma over-
expression of miR-30b/30d increases interleukin-10
synthesisbytargeting
GALNT7.20Interleukin-10 increases primary tumor
regulatory T cells and decreases
dendritic cells, and data also support an association
between miR-30d and regulatory T cell recruit-
ment.20Dendritic cells that express CD86 are often
regarded as both mature and immunogenic with the
capacity to prime T cells integral to the antitumor
immune response, but our study shows that CD86þ
dendritic cells increase the risk for sentinel lymph
node positivity. These ‘mature’ dendritic cells may
represent tolerogenic indoleamine 2,3-dioxygenase
(IDO)þ, CD123þplasmacytoid dendritic cells that
were found to be associated with poor prognosis
in melanoma15rather than immunogenic CD11cþ
conventional dendritic cells that were observed
to protect against sentinel lymph node meta-
stasis. CD86 can also be expressed on semi-mature
dendritic cells, which are tolerogenic.21
The apparent poor prognostic value of CD86þ
dendritic cells is likewise seen in the comparison
of the positive vs negative sentinel lymph node, in
which a significantly higher proportion of cells
expressing the dendritic cell maturation marker
CD86 is found in the positive sentinel lymph node.
Data again suggest that these ‘mature’ dendritic cells
are not of the conventional dendritic cell subset
as CD11cþcells are significantly decreased in the
positive sentinel lymph node compared with the
negative sentinel lymph node. The increased num-
ber of regulatory T cells in the positive sentinel
lymph node may instead have induced the preferen-
tial expansion of IDOþ, CD123þ
dendritic cells,22,23as was seen in a breast cancer
study where the presence of both Foxp3þand IDOþ
cells in the sentinel lymph node accurately dis-
criminated patients with a positive sentinel lymph
node from those with a negative sentinel lymph
node.24Our results, therefore, provide evidence
in support of the proposed positive feedback
loop between regulatory T cells and plasmacytoid
dendritic cells where regulatory T cells induce IDO
expression in plasmacytoid dendritic cells via
CTLA-4 signaling and IDO-expressing plasmacytoid
dendritic cells in turn induce regulatory T cell
differentiation,25which creates a microenvironment
conducive to tumor progression. The role of IDO-
expressing plasmacytoid dendritic cells in modulat-
ing the immunological status of the positive sentinel
lymph node is further supported by gene expression
data from a melanoma study that showed a trend
towards increased IDO levels in the positive sentinel
lymph node compared with non-sentinel nodes from
the same nodal basin.23Not only do IDO-expressing
plasmacytoiddendritic
progression,which is
the GalNActransferase
conventional
plasmacytoid
cells downregulatethe
immune response via regulatory T cells, but they
also cause a decrease in antitumor T cells. IDO
initiates the catabolism of the amino acid trypto-
phan, whose metabolites induce a stress response in
effector T cells resulting in cell-cycle arrest and an
increased susceptibility to Fas-mediated apoptosis.26
The selective expansion of regulatory T cells in
the positive sentinel lymph node also observed
in our study is consistent with findings from a study
in breast cancer where the proportion of Foxp3þ
cells in the sentinel lymph node of patients with
regional metastasis was shown to be significantly
higher than that of sentinel lymph node-negative
patients.24Previous studies evaluating the associa-
tion between Foxp3 positivity and nodal status in
melanoma and lung cancer have similarly shown a
significant increase in the frequency of regulatory
T cells in metastatic compared with tumor-free
draining lymph nodes.27,28
regional immunity in sentinel lymph nodes with
and without melanoma, however, revealed no signi-
ficant difference in the number of Foxp3þcells in
the positive sentinel lymph node compared with the
negative sentinel lymph node.29Despite the impor-
tance of reciprocal tumor-microenvironmental inter-
actions in melanoma progression, these authors
excluded areas infiltrated by melanoma cells in
their quantification of Foxp3þcells in the positive
sentinel lymph node as tumor cells may also stain
positive for Foxp3,30an issue addressed in our study
as regulatory Tcells were identified by the combina-
tion of Foxp3 positivity and characteristic lympho-
cytic morphology.
Sentinel lymph node positivity delineates a
group of melanoma patients at increased risk for
recurrence, and most of these patients undergo a
completion dissection, which is often followed by
systemic adjuvant therapy to lower this risk.
Immune markers can provide additional prognostic
information as our group has previously identified
an immune response gene expression signature that
improved the ability of the current American Joint
Committee on Cancer melanoma staging system to
predict clinical outcome.31In this study, we show
that the addition of regulatory T cell expression in
the sentinel lymph node similarly improves the
discriminative power of a recurrence risk assess-
ment model based on the American Joint Committee
on Cancer stage alone. The immune cell profile
of the sentinel lymph node not only provides
additional staging information, but it also has the
potential to guide adjuvant immunotherapeutic
decisions. Adjuvant therapies are associated with
considerable toxicity and currently, only interferon-
a-2b32is approved by the United States Food and
Drug Administration for use in high-risk melanoma
patients. Many other systemic adjuvant treatments,
such as different types of vaccine approaches
or immune modulation and more recently, anti-
CTLA-4 blockade, have been tested or are currently
under investigation. Vaccine antigens are often
A recent study of
Immune response in melanoma
MW Ma et al
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glycosylated, and regulatory T cells have been
shown to downregulate the expression of C-type
lectin receptors on dendritic cells that uptake these
carbohydrate-bearing antigens.33Regulatory T cell
depletion by biologics like denileukin diftitox
and cyclophosphamide would therefore boost the
immunological response to the vaccine as would
therapeutic agents that inhibit regulatory T cells.
Regulatory T cells together with IDO-expressing
plasmacytoid dendritic cells promote tumor pro-
gression by maintaining an immunosuppressive
microenvironment. As regulatory T cells constitu-
tively express CTLA-4 and IDO expression in
plasmacytoid dendritic
CTLA-4 signaling, anti-CTLA-4 monoclonal anti-
bodies would effectively break this positive feed-
back loop, and anti-CTLA-4 monoclonal antibodies
like ipilimumab have already demonstrated survival
benefit in metastatic melanoma patients34such that
it recently received Food and Drug Administration
approval for the treatment of unresectable stage III
and stage IV melanoma.
In conclusion, our study has shown that the
immune profile of the primary melanoma has
predictive value for sentinel lymph node positivity
and that the addition of primary tumor immune
markers as selection criteria for a sentinel lymph
node biopsy may aid in the identification of patients
with occult nodal metastasis. Data also suggest that
the immunological status of the sentinel lymph
node, the initial site of metastasis, provides infor-
mation for both staging and informed therapeutic
decisions that can potentially be used to restore an
effective antitumor immune response.
cells ismediated by
Acknowledgement
This work was supported by the Dr Alfred W Kopf
Research Grant Award from The Skin Cancer
Foundation (to FD), the National Cancer Institute
Cancer Center Support Grant (Grant No. 5 P30 CA
016087-27 to IO), and the Marc Jacobs Campaign to
support the Interdisciplinary Melanoma Coopera-
tive Group.
Disclosure/conflict of interest
The authors declare no conflict of interest.
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Supplementary Information accompanies the paper on Modern Pathology website (http://www.nature.com/
modpathol)
Immune response in melanoma
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