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The Journal of Clinical Endocrinology & Metabolism 87(9):4391– 4397
Copyright © 2002 by The Endocrine Society
doi: 10.1210/jc.2002-011262
Intense Expression of the B7-2 Antigen Presentation
Coactivator Is an Unfavorable Prognostic Indicator
for Differentiated Thyroid Carcinoma of Children
and Adolescents
RIMA SHAH, KEVIN BANKS, ANEETA PATEL, SHALINI DOGRA, RICHARD TERRELL,
PATRICIA A. POWERS, CYDNEY FENTON, CATHERINE A. DINAUER, R. MICHAEL TUTTLE,
GARY L. FRANCIS
AND
Department of Pediatrics, Uniformed Services University of the Health Sciences (R.S., K.B., A.P., S.D., R.T., P.A.P., C.F.,
C.A.D., G.L.F.), Bethesda, Maryland; Departments of Pediatrics (P.A.P., C.F., G.L.F.) and Clinical Investigation (C.A.D.),
Walter Reed Army Medical Center, Washington, D.C.; and Department of Endocrinology, Memorial Sloan Kettering Cancer
Center (R.M.T.), New York, New York
Previous observations suggest that an immune response
against thyroid carcinoma could be important for long-term
survival. We recently found that infiltration of thyroid carcinoma by proliferating lymphocytes is associated with improved disease-free survival, but the factors that control lymphocytic infiltration and proliferation are largely unknown.
We hypothesized that the antigen presentation coactivators
(B7-1 and B7-2), which are important in other immune-mediated thyroid diseases, might be important in lymphocytic infiltration of thyroid carcinoma. To test this, we determined
B7-1 and B7-2 expression by immunohistochemistry [absent
(grade 0) to intense (grade 3)] in 27 papillary (PTC) and 8
follicular (FTC) thyroid carcinomas and 9 benign thyroid lesions. B7-1 and B7-2 were expressed by the majority of PTC
and FTC (78% of PTC and 100% of FTC expressed B7-1; 88%
of PTC and 88% of FTC expressed B7-2). B7-1 expression
was more intense in PTC (1.4 ⴞ 0.2; P ⴝ 0.01) and FTC (2.6 ⴞ 0.2;
S
EVERAL OBSERVATIONS SUGGEST that an immune
response against differentiated thyroid carcinoma
could be important in long-term survival. Patients with papillary thyroid carcinoma (PTC) and lymphocytic thyroiditis
follow a more indolent clinical course than do patients with
PTC alone (1). Infiltration of PTC by tumor-associated lymphocytes has been associated with lower stages of disease at
diagnosis, and infiltration by proliferating lymphocytes has
been associated with improved disease-free survival (2, 3).
Despite the importance of these observations, the factors that
control lymphocytic infiltration and proliferation in thyroid
tumors remain largely unknown.
Antigen presentation by thyroid follicular cells is an important feature of other immune-mediated diseases of the
thyroid (4 – 6). Effective antigen presentation requires the
major histocompatibility class II complex and the antigen
presentation coactivators, B7-1 and B7-2 (7–9). The clinical
impact of these coactivators has been shown for patients with
Graves’ disease, in whom the majority of genetic propensity
Abbreviations: FTC, Follicular thyroid carcinoma; PTC, papillary thyroid carcinoma; MACIS, metastasis-age-completeness-of-resectioninvasion-size score; Tg, thyroglobulin.
P < 0.001) than in benign tumors (0.57 ⴞ 0.30) or presumably
normal adjacent thyroid (0.07 ⴞ 0.07) and was more intense in
carcinoma that contained lymphocytes (1.95 ⴞ 0.21) than in carcinoma that did not (1.08 ⴞ 0.26; P ⴝ 0.016). B7-2 expression was
of similar intensity in benign and malignant tumors (PTC, 1.6 ⴞ
0.2; FTC, 2.1 ⴞ 0.4; benign, 1.86 ⴞ 0.4), but was more intense than
in presumably normal adjacent thyroid (0.64 ⴞ 0.25; P < 0.013).
B7-2 expression also correlated with the number of tumor-associated lymphocytes per high power field (r ⴝ 0.38; P ⴝ 0.02).
Recurrence developed exclusively from tumors that expressed
B7-2, and intense B7-2 expression was associated with a reduced
probability of remission (P ⴝ 0.04). In conclusion, these data
support the hypothesis that the antigen presentation coactivators B7-1 and B7-2 may be important for lymphocytic infiltration
and the immune response against thyroid carcinoma. (J Clin
Endocrinol Metab 87: 4391– 4397, 2002)
for Graves’ disease has been linked to expression of the
receptor (CTLA-4) for the B7-2 coactivator (10 –13). Complete
functional profiles for these coactivators are not yet known.
However, B7-1 is increased in Hashimoto’s thyroiditis, but
not in Graves’ disease. B7-1 binds to the CD 28 T cell receptor,
where it generates a strong signal for T cell proliferation (7,
14). The B7-2 coactivator is increased in experimental thyroiditis and has been detected on intrathyroidal monocytes
and lymphocytes (15). B7-2 binds to the CTLA-4 receptor on
T cells, where it generates a negative growth signal (15).
Based on the central role of B7-1 and B7-2 coactivators
during antigen presentation, we hypothesized that alterations in B7-1 or B7-2 expression might be important in the
immune response against thyroid carcinoma. We tested this
by determining B7-1 and B7-2 expression in a group of PTC,
FTC, and benign lesions from children and adolescents. Our
data show that B7-1 and B7-2 coactivators are expressed by
the majority of PTC and FTC and are associated with the
presence of tumor-associated lymphocytes. Intense expression of B7-2 is associated with a reduced probability of remission and may also be associated with an increased recurrence risk.
4391
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J Clin Endocrinol Metab, September 2002, 87(9):4391– 4397
Subjects and Methods
Patients
The automated centralized tumor registry of the Department of Defense was searched to identify all patients with differentiated thyroid
carcinoma who were 21 yr of age or younger at the time of diagnosis.
A computerized database that includes demographic features, tumor
characteristics, surgical treatment, adjunctive therapy, and clinical outcome was generated from these data and used in previous publications
(16 –18). The extent of disease at diagnosis was classified according to the
system of DeGroot et al. (19) and the metastasis-age-completeness-ofresection-invasion-size score (MACIS). According to DeGroot et al., class
1 disease was confined to the thyroid gland, class 2 involved the regional
lymph nodes, class 3 either extended beyond the capsule or was inadequately resected, and class 4 had distant metastasis (20). Because all
patients were less than 39 yr of age, MACIS scores were calculated as
3.1 ⫹ (size ⫻ 0.3) ⫹ 1 (if incomplete resection) ⫹ 3 (if distant metastasis)
(19). As in previous studies, recurrence was defined as the appearance
of new disease (identified by radioactive iodine scan or biopsy) in any
patient who had been free of disease (no disease palpable or identified
by radioactive iodine scan) for a period of 4 months after initial therapy
(16). Serum thyroglobulin (Tg) values were determined in contemporary
patients (normal range, 3– 40 ng/ml; University of Southern California
Clinical Laboratories, Los Angeles, CA). The clinical details for some of
the patients in this group have been previously published (16 –18).
Formalin-fixed, paraffin-embedded, archival tumor blocks corresponding to 27 PTC, 8 FTC, 9 benign thyroid lesions (5 benign follicular
adenomas, 1 Graves’ disease, 2 multinodular goiters, and 1 Hashimoto’s
thyroiditis), and 1 normal thyroid were available for study. For analysis,
the benign lesions were separated into two categories (benign tumors
and autoimmune lesions) and compared with PTC and FTC. Sufficient
material was available to examine the intensity of B7-1 and B7-2 staining
on regions of presumably normal thyroid tissue adjacent to 14 PTC and
FTC. These are included in the text and figures as normal thyroid. The
only truly normal thyroid is identified throughout.
Immunohistochemistry
Sections from original, formalin-fixed, paraffin-embedded, archival
tissue blocks were sectioned and stained with hematoxylin and eosin to
confirm the diagnosis (21). The sections immediately adjacent (5 ␮m)
were deparaffinized with xylene and rehydrated through a series of
graded alcohol solutions. Endogenous peroxidase was inactivated (3%
H2O2, 30 min), and antigen was retrieved in citrate buffer (pH 6.0, 30 min,
100 C, steamer). For determination of B7-1, tissue sections were sequentially incubated with primary monoclonal B7-1 antibody (1:50, 37 C, 1 h;
catalog no. 1634, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by biotinylated secondary antibody (1:100, 37 C, 30 min) using the
Ventana Nexes automated immunostainer (Ventana Medical Systems,
Tucson, AZ). This was then coupled with a streptavidin conjugateenzyme complex (30 min, 37 C; Vector Laboratories, Inc., Burlingame,
CA). The substrate chromogen (diaminobenzidine, 10 min, room temperature) and hematoxylin counterstain (30 sec) were manually added.
Sections of tonsil were used as the positive control, and PBS was substituted for the primary and secondary antibodies and used as the
negative control. The intensity of B7-1 staining was based on the intensity of chromogen developed throughout the majority of each tumor.
Staining intensity was determined by two blinded, independent examiners and was graded as follows: 0 ⫽ absent, 1 ⫽ minimal, 2 ⫽ moderate,
and 3 ⫽ intense. The interobserver agreement was greater than 95%, and
the few discordant slides were graded by a third examiner. The two
scores in agreement were then used as the final intensity grade. Identical
procedures were used for determination of B7-2 expression, except that
the primary antibody was directed against B7-2 (catalog no. SC 1635,
Santa Cruz Biotechnology, Inc.).
We previously stained these same tumors for the number of lymphocytes (leukocyte common antigen) and number of proliferating lymphocytes per high power field [cells positive for both proliferating cell
nuclear antigen (Ki-67) and leukocyte common antigen] (3). The methods and results were previously described (3, 22). In the current study
B7-1 and B7-2 expression was compared with the number of lymphocytes and proliferating lymphocytes in each tumor.
Shah et al. • Expression of B7-2 in Prognosis of PTC and FTC
Data analysis and statistical comparisons
The intensity of B7-1 and B7-2 expression for PTC, FTC, and benign
lesions was then compared and correlated with the demographic features, histologic variant, focality of the tumor, size of the tumor, extent
of disease at diagnosis (class 1– 4), MACIS score, and clinical outcome.
Statistical analyses were performed using SPSS for Windows 95 (version
7.5, SPSS, Inc., Chicago, IL). The intensities of B7-1 and B7-2 expression
were compared by ANOVA, correlations were performed using Pearson
correlation, and recurrence-free survival was calculated using KaplanMeier survival curves with log-rank comparison. Nonparametric analyses were performed using either the ␹2 or Fisher’s exact test as indicated. Similar statistical analyses were used to correlate the number of
proliferating lymphocytes per high power field for PTC and FTC with
B7-1 and B7-2 expression.
Results
The specificity of B7-1 and B7-2 staining were confirmed
using human tonsil as a positive control and PBS for the
negative control. Diffuse, intense staining was seen in the
tonsil and was completely abolished by substitution of PBS
for the primary or secondary antibody. The length of time
each tumor block had been stored had no effect on the intensity of B7-1 or B7-2 staining (B7-1: r ⫽ 0.01; P ⫽ 0.93; B7-2:
r ⫽ 0.04; P ⫽ 0.81). Representative staining of each intensity
is shown in Fig. 1. Of note, specific staining for B7-1 and B7-2
was detected in thyroid follicular cells and was of equal
intensity in areas adjacent to infiltrating lymphocytes as well
as in areas that were remote from infiltrating lymphocytes.
The demographic features of the patients with PTC and
FTC in this study are similar to those of the larger series
previously reported by our group (16). For patients with
PTC, the average age was 16.1 ⫾ 4.4 yr (range, 6 –21 yr), the
mean tumor size was 1.8 ⫾ 1.2 cm (range, 0.5–5.5 cm), the
average MACIS score was 3.95 ⫾ 0.95 (range, 3.25–7.39), and
the average follow-up was 56 ⫾ 44 months (range, 0 –169
months). For patients with FTC, the average age was 17 ⫾ 2.4
yr (range, 13–20 yr), the mean tumor size was 2.2 ⫾ 1.3 cm
(range, 0.2– 4.0 cm), and the average follow-up was 59 ⫾ 55
months (range, 18 –168 months). Patients with PTC and FTC
were stratified according to the expression of B7-1 (Table 1)
and B7-2 (Table 2). The demographic features and treatment
received by each group were similar. Except for one patient
who underwent lobectomy, all patients with PTC underwent
total or subtotal thyroidectomy. All patients with FTC were
treated with total thyroidectomy, except for one who underwent subtotal thyroidectomy. Radioactive iodine was administered to 59% of patients with PTC and 63% of patients
with FTC.
Overall, B7-1 was detected in a larger proportion of malignant tumors (78% of PTC and 100% of FTC) than presumably normal adjacent thyroid (1 of 14, 7%; P ⬍ 0.001). The
proportion of benign thyroid tumors with detectable B7-1
expression (4 of 7, 57%) was not significantly different from
that found in malignant tumors (P ⫽ 0.26 vs. PTC and P ⫽
0.077 vs. FTC), but the number of samples may have been too
small to achieve statistical significance. B7-1 staining (Fig.
2A) was more intense for PTC (1.4 ⫾ 0.2; P ⫽ 0.035) and FTC
(2.6 ⫾ 0.2; P ⬍ 0.001) than for benign tumors (0.57 ⫾ 0.30) or
presumably normal adjacent thyroid (0.07 ⫾ 0.07; P ⱕ 0.01).
The only truly normal thyroid in this study had no B7-1
staining.
Shah et al. • Expression of B7-2 in Prognosis of PTC and FTC
J Clin Endocrinol Metab, September 2002, 87(9):4391– 4397 4393
FIG. 1. Representative immunostaining for B7-1 and B7-2. B7-1 staining is absent (A), minimal (B), moderate (C), or intense (D). B7-2 staining
is absent (E), minimal (F), moderate (G), or intense (H). All photomicrographs are shown at ⫻200 magnification.
TABLE 1. Clinical features and B7-1 expression for PTC and FTC
PTC
Female/male
Age (yr ⫾ SEM)
Tumor size (cm ⫾ SEM)
MACIS (mean ⫾ SEM)
Class
1
2
3
4
Unifocal/multifocal
Remission Y (%)
Persistent Y (%)
Recur Y (%)
Time to recur (months ⫾ SEM)
Follow-up (months ⫾ SEM)
All
(n ⫽ 27)
Grade 0
(n ⫽ 6)
Grade 1
(n ⫽ 6)
Grade 2
(n ⫽ 13)
Grade 3
(n ⫽ 2)
P
20/7
16 ⫾ 4
1.8 ⫾ 1.2
3.9 ⫾ 0.9
5/1
15 ⫾ 4.7
2.2 ⫾ 0.8
3.8 ⫾ 0.24
5/1
16.6 ⫾ 5.6
1.7 ⫾ 1.4
4.8 ⫾ 1.7
9/4
16.5 ⫾ 4.1
1.4 ⫾ 0.7
3.5 ⫾ 0.2
1/1
15 ⫾ 7.8
4.0 ⫾ 2.1
4.8 ⫾ 0.1
0.73
0.76
0.68
0.48
2
1
1
2
2/4
3 (50%)
3 (50%)
2 (33%)
11.5 ⫾ 7.8
91 ⫾ 16
9
4
0
0
8/5
9 (69%)
4 (31%)
2 (15%)
11.5 ⫾ 0.7
47 ⫾ 11
0
1
1
0
0/2
1 (50%)
1 (50%)
0
N/A
1.5 ⫾ 1.5
0.79
15
5
2
2
13/14
15 (55%)
10 (37%)
5 (19%)
22.6 ⫾ 25
56 ⫾ 44
4
2
0
0
3/3
3 (50%)
2 (33%)
1 (17%)
67 (n ⫽ 1)
60 ⫾ 18
0.34
0.79
0.84
0.77
ns
0.04
FTC
All
(n ⫽ 8)
Female/male
Age (yr ⫾ SEM)
Tumor size (cm ⫾ SEM)
Recur Y (%)
Time to recur (months ⫾ SEM)
Follow-up (months ⫾ SEM)
Grade 0
(n ⫽ 0)
6/2
17 ⫾ 2.4
2.2 ⫾ 1.3
1 (12%)
9 (n ⫽ 1)
59 ⫾ 55
B7-2 was detected in a similar majority of benign and
malignant lesions (88% of PTC, 88% of FTC, and 100% of
benign lesions), but was detected in a smaller proportion of
presumably normal adjacent thyroids (6 of 14, 43%; P ⫽ 0.04
vs. FTC; P ⫽ 0.002 vs. PTC). The intensity of B7-2 staining (Fig.
2B) was similar for benign and malignant tumors (PTC, 1.6 ⫾
0.2; FTC, 2.1 ⫾ 0.4; benign, 1.86 ⫾ 0.4), but significantly
greater than in presumably normal adjacent thyroid (0.64 ⫾
0.25; P ⱕ 0.013). The only truly normal thyroid in this study
had intense (grade 3) B7-2 staining.
Several of the tumors in this study had been previously
examined for the presence of tumor-associated lymphocytes
(3). In our previous study we found that tumors with the
Grade 1
(n ⫽ 0)
Grade 2
(n ⫽ 3)
Grade 3
(n ⫽ 5)
P
2/1
17.3 ⫾ 3.8
1.4 ⫾ 1.3
0
3/2
16.7 ⫾ 0.6
2.9 ⫾ 1.0
1 (20%)
9
45 ⫾ 8
0.71
0.78
0.19
0.63
73 ⫾ 47
0.59
lowest number of proliferating lymphocytes per high power
field had the greatest risk for recurrence. However, there was
no correlation between the total number of lymphocytes per
high power field and the risk of recurrence (3). Staining for
B7-1 and B7-2 was compared with the number of lymphocytes and the number of proliferating lymphocytes per high
power field. In addition to having more intense B7-1 staining,
malignant tumors had more numerous lymphocytes per
high power field (31 ⫾ 8.5 for PTC, P ⫽ 0.004; 6.4 ⫾ 2.8 for
FTC, P ⫽ 0.32) and more numerous proliferating lymphocytes per high power field (7.0 ⫾ 3.8 for PTC, P ⫽ 0.08; 6.0 ⫾
3.0 for FTC, P ⫽ 0.08) compared with benign tumors (lymphocytes, 2.5 ⫾ 2.5/high power field; proliferating lympho-
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J Clin Endocrinol Metab, September 2002, 87(9):4391– 4397
Shah et al. • Expression of B7-2 in Prognosis of PTC and FTC
TABLE 2. Clinical features and B7-2 expression for PTC and FTC
PTC
Female/male
Age (yr ⫾ SEM)
Tumor size (cm ⫾ SEM)
MACIS (mean ⫾ SEM)
Class
1
2
3
4
Unifocal/multifocal
Remission Y (%)
Persistent Y (%)
Recur Y (%)
Time to recur (months ⫾ SEM)
Follow-up (months ⫾ SEM)
All
(n ⫽ 25)
Grade 0
(n ⫽ 3)
Grade 1
(n ⫽ 10)
Grade 2
(n ⫽ 5)
Grade 3
(n ⫽ 7)
P
18/7
16.6 ⫾ 4
1.8 ⫾ 1.2
4.0 ⫾ 1.0
1/2
17.7 ⫾ 3.2
0.7 ⫾ 0.3
4.3 ⫾ 1.7
8/2
17 ⫾ 4.5
1.8 ⫾ 1.1
3.9 ⫾ 1.2
4/1
14.6 ⫾ 4.5
1.8 ⫾ 0.66
3.6 ⫾ 0.2
5/2
16.9 ⫾ 3.6
2.2 ⫾ 1.6
4.1 ⫾ 0.7
0.49
0.65
0.55
0.78
14
7
2
2
12/13
14 (56%)
10 (40%)
4 (16%)
11.5 ⫾ 4.5
56 ⫾ 44
1
1
0
1
0/3
2 (67%)
1 (33%)
0
7
2
0
1
6/4
6 (60%)
4 (40%)
1 (10%)
6
81 ⫾ 14
4
1
0
0
4/1
5 (100%)
0
1 (20%)
11
33 ⫾ 9.9
2
3
2
0
2/5
1 (14%)
5 (71%)
2 (29%)
14.5 ⫾ 3.5
39 ⫾ 16
0.60
Grade 2
(n ⫽ 0)
Grade 3
(n ⫽ 5)
P
4/1
15.7 ⫾ 2.3
1.9 ⫾ 0.7
1 (20%)
81 ⫾ 43
0.71
0.12
0.70
0.71
0.66
59 ⫾ 27
0.09
0.03
0.07
0.11
ns
0.12
FTC
Female/male
Age (yr ⫾ SEM)
Tumor size (cm ⫾ SEM)
Recur Y (%)
Follow-up (months ⫾ SEM)
All
(n ⫽ 8)
Grade 0
(n ⫽ 1)
Grade 1
(n ⫽ 2)
6/2
17 ⫾ 2.4
2.2 ⫾ 1.3
1 (12%)
59 ⫾ 55
1/0
16
4.0
0
60
2/0
19.5 ⫾ 0.7
1.5 ⫾ 1.8
0
26 ⫾ 8
cytes, 0.1 ⫾ 0.07/high power field). When PTC and FTC were
stratified into groups that either did or did not contain lymphocytes, carcinoma that contained lymphocytes had significantly more intense B7-1 staining (1.95 ⫾ 0.21) than
carcinoma that did not contain lymphocytes (1.08 ⫾ 0.26;
P ⫽ 0.016).
As shown in Fig. 3, there was a significant correlation
between the overall intensity of B7-2 staining and the number
of lymphocytes per high power field (r ⫽ 0.38; P ⫽ 0.02). This
relationship was even more significant when the analysis
was confined solely to PTC (r ⫽ 0.66; P ⫽ 0.012; data not
shown). There was no overall relationship between the intensity of B7-2 staining and the number of proliferating lymphocytes per high power field (r ⫽ 0.01; P ⫽ 0.93). However,
when PTC and FTC were stratified into carcinoma with moderate to intense B7-2 staining and compared against carcinoma with absent to minimal B7-2 staining, almost twice as
many proliferating lymphocytes were found per high power
field in the carcinoma with absent to minimal B7-2 staining
(10 ⫾ 7.6/high power field) compared with PTC with moderate to intense B7-2 staining (4.9 ⫾ 2.4/high power field; P ⫽
0.53). These observations are complex, but suggest that B7-1
and B7-2 expression could be associated with lymphocytic
infiltration and that intense B7-2 expression might inhibit
lymphocyte proliferation.
Clinical outcomes were compared for PTC and FTC stratified according to B7-1 and B7-2 staining. The risk of recurrent disease was similar for tumors that did or did not express B7-1 (B7-1 absent, 17% recurrence; B7-1 minimal, 33%
recurrence; B7-1 moderate, 13% recurrence; B7-1 intense, 17%
recurrence). In addition, there was no significant relationship
between B7-1 staining and disease-free survival (P ⫽ 0.79;
data not shown), the probability of remission at the last visit
(P ⫽ 0.79), or the risk of persistent disease (P ⫽ 0.84).
In contrast, recurrence developed exclusively among tumors with detectable B7-2 staining, and the risk of recurrence
was increased in tumors with more intense B7-2 staining
(B7-2 absent, no recurrence; B7-2 minimal, 8% recurrence;
B7-2 moderate, 20% recurrence; B7-2 intense, 30% recurrence). However, the number of recurrent tumors was too
small to achieve statistical significance (P ⫽ 0.10, analysis of
trend). The last known status of each patient was significantly correlated with B7-2 staining. PTC and FTC with more
intense B7-2 staining were less likely to be in remission (P ⫽
0.04, linear by linear association; P ⫽ 0.03 for PTC only) and
more likely to have persistent disease (P ⫽ 0.07).
Discussion
Previous observations suggest that an immune response
against thyroid carcinoma could be important in long term
survival (1–3). In a recent study, infiltration of differentiated
thyroid carcinoma by proliferating lymphocytes was associated with improved disease-free survival (3). However, the
factors that control lymphocytic infiltration and proliferation
remain unknown. The antigen presentation coactivators B7-1
and B7-2 have a critical role in other immune-mediated thyroid diseases (4 –9). We therefore examined B7-1 and B7-2
coactivator expression in a group of benign and malignant
thyroid lesions. To our knowledge, our data are the first to
examine the relationships between B7-1 and B7-2 coactivator
expression and clinical outcomes for thyroid carcinoma.
The data show that B7-1 and B7-2 can be detected by
immunohistochemistry in a greater proportion of benign and
malignant thyroid lesions compared with presumably normal adjacent thyroid. B7-1 was detected in a higher proportion of malignant tumors and was also more intense. B7-1
expression was absent from the only truly normal thyroid in
Shah et al. • Expression of B7-2 in Prognosis of PTC and FTC
J Clin Endocrinol Metab, September 2002, 87(9):4391– 4397 4395
FIG. 2. Comparison of B7-1 and B7-2 expression in benign, malignant, and presumably normal adjacent thyroid. A, The expression of B7-1 was significantly greater
in PTC and FTC compared with benign lesions or presumably normal thyroid. Data are presented as the
mean intensity ⫾ SEM, with significance determined by
ANOVA. B, The expression of B7-2 was similar in PTC,
FTC, and benign lesions, but significantly greater than
in presumably normal thyroid. Data are presented as
the mean intensity ⫾ SEM, with significance determined
by ANOVA.
this study. Malignant tumors contained more numerous
lymphocytes and proliferating lymphocytes than did benign
tumors. Taken together, these data suggest that B7-1 expression could be related to the presence of tumor-associated
lymphocytes. It is not clear from these data whether B7-1
expression promotes lymphocytic infiltration or whether
lymphocytic infiltration induces B7-1 expression. Matsuoka
et al. (15) previously identified B7-1 and B7-2 coactivators on
the surface of tumor-infiltrating lymphocytes and monocytes. They proposed augmentation of B7-1 and B7-2 expression by an interaction between thyroid follicular cells and
true antigen-presenting cells. In direct support of this hy-
pothesis, we found that PTC and FTC containing lymphocytes had more intense B7-1 staining compared with PTC and
FTC that did not contain lymphocytes.
B7-2 was detected in a similar majority of benign and
malignant thyroid tumors and was more frequently detected
than in presumably normal adjacent thyroid. The intensity of
B7-2 staining was similar for benign and malignant tumors
and was of greater intensity among all tumors than in sections of presumably normal adjacent thyroid. The only truly
normal thyroid in this study demonstrated intense B7-2
staining. The reason for this is not clear from our study. It is
possible that the presumably normal adjacent thyroid tissues
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J Clin Endocrinol Metab, September 2002, 87(9):4391– 4397
Shah et al. • Expression of B7-2 in Prognosis of PTC and FTC
FIG. 3. Correlation between B7-2 expression and the
number of lymphocytes per high power field. The intensity of B7-2 expression was correlated with the number
of lymphocytes per high power field and is shown for all
benign and malignant lesions. There was a significant
correlation (Pearson correlation) between B7-2 expression and the number of lymphocytes per high power field
that was even more significant when the analysis was
confined solely to PTC (r ⫽ 0.66; P ⫽ 0.012; data not
shown).
might not be entirely normal. These samples were obtained
from the immediate vicinity of malignant lesions and might
contain genetic alterations that predispose to malignant
transformation. It is also possible that the one normal thyroid
in our study might not be representative of normal thyroid.
We are unaware of any clinical history for this patient, and
it is possible he/she might have had a nonthyroidal condition that resulted in generalized up-regulation of B7-2 expression. Additional studies of B7-1 and B7-2 expression in
the normal thyroid are clearly warranted; however, our data
reveal important relationships between B7-2 staining and
clinical outcomes for patients with PTC and FTC.
There was a significant correlation between the intensity
of B7-2 staining and the number of lymphocytes per high
power field. This observation is consistent with either B7-2
promotion of lymphocyte infiltration or induction of B7-2
expression by tumor-associated lymphocytes. It is believed
that the B7-2 signal, mediated by the CTLA-4 receptor, inhibits T cell proliferation (13). Our data are consistent with
this theory in that tumors with more intense B7-2 staining
contained fewer proliferating lymphocytes per high power
field compared with PTC with less intense B7-2 expression.
The latter difference, however, was not statistically significant (P ⫽ 0.53).
Recurrent disease developed exclusively from PTC and
FTC with detectable B7-2 staining. Furthermore, there was a
suggestion that the intensity of B7-2 staining was directly
related to the risk of recurrence (0% recurrence with absent
B7-2, 8% recurrence with minimal B7-2, 20% recurrence with
moderate B7-2, and 30% recurrence with intense B7-2 expression; P ⫽ 0.10) and was inversely related to disease-free
survival (P ⫽ 0.35, log rank comparison). The small sample
size limits the power of these observations and may be responsible for the fact that neither observation achieved statistical significance. When the last known clinical status for
each patient was correlated with B7-2 staining, patients with
intense B7-2 staining were less likely to be in remission (P ⫽
0.04, linear by linear association) and more likely to have
persistent disease (P ⫽ 0.07). The average length of follow-up
was similar for patients with all grades of B7-2 staining.
Taken together, these data are consistent with the hypothesis
that B7-2 expression could inhibit T cell proliferation, leading
to an increased risk for persistent or recurrent disease.
Our observations are limited by the retrospective nature of
the treatment and follow-up data. Serum Tg levels were
available only for the contemporary patients. Thyroid hormone withdrawal was used in contemporary patients to
stimulate Tg production. Stimulated Tg levels were less than
2 ng/ml in all contemporary patients classified as free of
disease. Unfortunately, serum Tg levels were not available
for the historical patients, in whom recurrence was defined
by computerized tomographic images, radioactive iodine
scan, and clinical examination. It is possible that some of
these patients could have had detectable serum Tg and
would have been erroneously classified as free of disease.
However, treatment for these scan-negative, Tg-positive patients remains controversial (22).
A previous study by Lahat et al. (7) used flow cytometry
and determined that the B7 surface molecules were absent
from human thyroid cancer cell lines and could not be induced by interferon. We do not have a complete explanation
for the difference between our findings and those of Lahat et
al. (7). It is possible that B7-1 and B7-2 coactivators are not
expressed in tissue culture, but are expressed in situ. This
could explain why B7-1 and B7-2 were detected in the majority of tumors in our study, whereas Lahat et al. (7) failed
to detect B7-1 or B7-2 expression on cultured thyroid cells.
Matsuoka et al. (15) proposed augmentation of B7-1 and B7-2
expression by an interaction between thyroid follicular cells
and true antigen-presenting cells. Such an interaction could
explain B7-1 and B7-2 expression in situ, but not in culture.
It is also possible that the difference between our studies
Shah et al. • Expression of B7-2 in Prognosis of PTC and FTC
could relate to the level of differentiation of the tumors used
in each study. We examined only well differentiated thyroid
carcinoma from children and adolescents, for whom longterm survival is excellent. The study by Lahat et al. (7) determined B7-1 and B7-2 expression using the papillary
carcinoma-derived cell line (NPA), two follicular carcinomaderived cell lines (MRO and WRO), and the anaplastic carcinoma cell line (ARO). Only the NPA cell line retains a
majority of differentiated thyroid functions. The MRO, WRO,
and ARO cells lines are either dedifferentiated or frankly
anaplastic. Had we included poorly differentiated or anaplastic tumors in our study, they might not have expressed
B7-1 or B7-2.
In conclusion, our data show that the B7-1 and B7-2 coactivators are expressed by differentiated thyroid carcinoma
of children and adolescents and are associated with the presence of lymphocytic infiltration. B7-1 staining is more intense, and the proportion of tumors that express B7-1 is
greater among malignant (PTC and FTC) tumors compared
with presumably normal thyroid. More intense B7-2 staining
is found among malignant tumors (PTC and FTC) with fewer
proliferating lymphocytes and is associated with a lower
probability of remission and an increased risk for recurrent
disease. Additional study is required to validate these findings on a larger and more diverse patient population; however, the data suggest the possibility that B7-1 and B7-2
coactivators could be important in the immune response
against thyroid carcinoma.
J Clin Endocrinol Metab, September 2002, 87(9):4391– 4397 4397
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Acknowledgments
This study received prior approval from the Human Use Committee,
Department of Clinical Investigation, Walter Reed Army Medical
Center.
15.
Received August 2, 2001. Accepted June 7, 2002.
Address all correspondence and requests for reprints to: Gary L.
Francis, M.D., Department of Pediatrics, Uniformed Services University
of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland
20814.
This work was supported by an intramural research grant (WU 006501) from the Department of Clinical Investigation, Walter Reed Army
Medical Center. The opinions or assertions contained herein are the
private views of the authors and are not to be construed as official or as
reflecting the opinions of the Uniformed Services University of the
Health Sciences, the Department of the Army, or the Department of
Defense.
16.
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